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In vivo selection of RNAs that localize in the nucleus

In vivo selection of RNAs that localize in the nucleus The EMBO Journal Vol.16 No.4 pp.793–806, 1997 1992; Izaurralde and Mattaj, 1992, 1995). Other nuclear Christian Grimm , Elsebet Lund and RNAs, like the small nucleolar RNAs (snoRNAs) and U6 James E.Dahlberg snRNA are not exported to the cytoplasm, but are retained Department of Biomolecular Chemistry, 1300 University Avenue, in the nucleus (Vankan et al., 1990; Terns and Dahlberg, University of Wisconsin, Madison, WI 53706, USA 1994; Boelens et al., 1995; Terns et al., 1995). Present address: Department of Ophthalmology, University Hospital- Several pathways exist for nuclear import of RNA Zu¨rich, 8091 Zu¨rich, Switzerland (Fischer et al., 1991; Michaud and Goldfarb, 1992). Corresponding author Efficient import of most spliceosomal snRNAs into nuclei, as snRNPs, requires two cis-acting elements: a binding Nuclear localization of an RNA is affected by cis- site for the Sm proteins and a trimethylguanosine cap acting elements (NLEs) that lead to nuclear import or structure (reviewed in Nigg et al., 1991; Izaurralde and retention or to blockage of export from the nucleus. Mattaj, 1992). In contrast, U6 snRNA (Hamm and Mattaj, To identify such elements, we selected and analyzed 1989) and 5S rRNA (Allison et al., 1993) lack both of transcripts that localized in the nuclei of Xenopus laevis these signals, but nevertheless can be imported into nuclei oocytes. The RNAs were isolated from a collection of of Xenopus oocytes, bound to proteins that contain nuclear m G-capped RNAs in which a combinatorial library localization signals (NLSs) (Fischer et al., 1991). (n  20) of sequences had been inserted. One class of Likewise, several distinct pathways are likely to exist selected RNAs (Sm ) had a consensus Sm binding also for RNA export, as demonstrated by the lack of site (AAUUUUUGG) and bound Sm proteins in the competition in this process between different classes cytoplasm; these RNAs resembled small nuclear RNAs of RNA molecules (Jarmolowski et al., 1994; Pokrywka like U1 and U5 RNAs in their bi-directional nucleo– and Goldfarb, 1995) and by the differential inhibition of cytoplasmic transport and their 5-cap hyper- export by various inhibitors of NPC function (E.Lund methylation. Another class, Sm RNAs, contained and J.E.Dahlberg, in preparation; Powers et al., 1997). sequences that masked the m G-caps of the RNAs However, several RNAs also share aspects of common and promoted interaction with La protein. These export pathways. For example, saturation of the Rev- RNAs were retained within nuclei after nuclear injec- mediated export of RRE-containing RNAs (Fischer et al., tion and were imported when injected into the cyto- 1994) affects export of pre-snRNAs and 5S RNA, but not plasm. Their nuclear import and retention were that of mRNAs and tRNAs (Fischer et al., 1995). independent of a 5-cap, required an imperfect double- The monomethylated cap structure of pre-snRNAs and stranded stem near the 5 end, and depended on mRNAs facilitates export of these RNAs to the cytoplasm interaction with La protein. Import of the Sm RNAs, (Hamm and Mattaj, 1990; Terns et al., 1993; Jarmolowski while using the import pathway of proteins, was distinct et al., 1994). This m G-cap is recognized by the proteins from that of U6 RNA. of the cap binding complex (CBC) which mediates export, Keywords: La protein/nuclear localization elements at least of snRNAs (Izaurralde et al., 1995; reviewed in (NLEs)/RNA transport/selection of RNAs in vivo/Sm Go¨rlich and Mattaj, 1996). Other structural elements of consensus site these RNAs are also important for export (Hamm and Mattaj, 1990; Eckner et al., 1991; Terns et al., 1993; Jarmolowski et al., 1994). Several RNA binding proteins have been implicated in the export of different classes of Introduction RNAs, but only CBP20 and CBP80 (the proteins of CBC; Izaurralde et al., 1995), and the Rev protein (Fischer et al., The distribution of RNAs within various sub-cellular 1995), have been shown to promote export of bound RNA compartments results from a balance of export, import (reviewed in Izaurralde and Mattaj, 1995; Go¨rlich and and retention. In several instances, cis-acting sequences Mattaj, 1996). in the RNA and trans-acting cellular factors have been The lectin wheat germ agglutinin (WGA), which binds shown to contribute to these processes. However, relatively to N-acetylglucosamine residues of NPC proteins, is an few of the actual proteins and RNA signals are known. effective inhibitor of many types of nucleo–cytoplasmic Intracellular RNA transport is an active process that often transport (reviewed by Forbes, 1992). Import of most involves translocation of the RNAs (or RNPs) across the proteins carrying an NLS is inhibited by WGA treatment nuclear envelope, through the nuclear pore complexes (NPCs; Davis, 1995). Whereas most RNAs (mRNA, tRNA (Finlay et al., 1987; Dabauvalle et al., 1988) as is the and scRNA) are transported unidirectionally from the import of U6 RNA, which apparently occurs by the same nucleus to the cytoplasm, the precursors of many small pathway (Fischer et al., 1991; this study). In contrast, nuclear RNAs (pre-snRNAs) and 5S ribosomal RNA are import of snRNPs containing Sm proteins is relatively translocated through the pores in both directions (Zapp, insensitive to the lectin. Export of most RNAs is inhibited © Oxford University Press 793 C.Grimm, E.Lund and J.E.Dahlberg by WGA (E.Lund and J.E.Dahlberg, manuscript in pre- paration), but some of these effects may be secondary to inhibition of import of proteins that are required as carriers. Several mechanisms could account for the localization of RNAs in nuclei. RNAs may contain sequences that lead to their retention within the nucleus or to their import from the cytoplasm, as has been demonstrated for various classes of snRNAs. Examples include a sequence element common to most snoRNAs (box D), that is essential for nuclear retention of U8 snoRNA (Terns et al., 1995), and the binding site for Sm proteins of the spliceosomal RNAs U1, U2, U4 and U5, which promotes import of these molecules into the nucleus. Alternatively, structures or sequences that ordinarily would direct export to the cytoplasm may either be absent or masked in other RNAs that localize in nuclei. Here we define the term nuclear localization element (NLE) as any cis-acting RNA sequence or structural feature that promotes localization of the RNA in the nucleus. In this study we developed and used an iterative selection to identify signals and mechanisms that contribute to the localization of RNAs within nuclei. At least two classes of molecules were isolated that had the capacity to be localized within nuclei. One class of RNAs contains a consensus sequence for the Sm binding site (AAU- UUUUGG) that promotes nuclear localization of snRNAs; isolation of this expected class of molecules validated the method. Another class contains a structural motif that participates in nuclear localization of Sm RNAs. Inter- action of this structure with La protein appears to promote Fig. 1. Selection of RNAs with nuclear localization signals. retention of the RNA in the nucleus by masking the 5- (A) Structure of the RNA used for the selection of NLEs. Fixed cap, which otherwise acts as an export signal; furthermore, nucleotide sequences from the 5 and 3 ends of U1 snRNA are in by binding to La protein in the cytoplasm the RNA can upper case letters, and nucleotides in lower case letters indicate be imported in an NLS-dependent fashion. changes in the U1 sequences that were made to ensure efficient in vitro transcription by T7 RNA polymerase (addition of Gs at the 5 end) and increased in vivo stability of the RNA (A to C change in Results the 5 stem). N20 represents the 20 nucleotide randomized sequence. Arrows indicate the endpoints of the primers used for reverse In vivo selection of RNA elements that promote transcription and PCR amplification of the RNAs. (B) Enrichment of nuclear localization the pools for RNAs that localize in the nucleus. The percentages of RNAs that localized in the nucleus at 20–24 h after nuclear injection To select for NLEs (both nuclear import and nuclear were determined by PhosphorImager analysis of gels run after each retention signals), we used a short derivative of U1 round of selection and were expressed as [N/(NC)]100. Thick snRNA as the carrier molecule for a 20 nucleotide long, – lines, experimental RNA pools; thin lines, control U1Sm , U2 and U3 randomized sequence (N20; Figure 1A; see also Grimm RNAs that were exported to the cytoplasm, exported and imported back into the nucleus and retained in the nucleus, respectively. et al., 1995). Since the carrier moiety of this in vitro Sm and Sm pools of RNAs were generated at rounds 4 and 8 transcribed RNA contains a strong nuclear export signal (circles) by immunoprecipitation of nuclear extracts with anti-Sm in the form of the m G-cap (Hamm and Mattaj, 1990; antibodies; the RNAs in the precipitate (Sm ) and supernatant (Sm ) Izaurralde et al., 1995) but no nuclear import signal, most fractions were injected separately after round 4. of the stable molecules of the starting pool localized in the cytoplasm within 20 h after they had been injected expected, one class of the molecules we isolate should into nuclei (Figure 1B; see also Figure 5A, top panel). contain Sm protein binding sites. To distinguish between However, some RNAs received a sequence through the molecules that contain such Sm binding sites and mole- N20 insert that caused nuclear localization of the RNA, cules with other NLEs, we used anti-Sm antibodies to and therefore could be isolated from nuclei and amplified precipitate RNAs bound to Sm proteins (at rounds 4 and through multiple rounds of selection. The percentage of 8, as indicated by circles, Figure 1B). The resulting RNAs in the experimental RNA pool that localized in the Sm and Sm RNA pools were treated separately after nucleus increased with each round, indicating a gradual round 4. After 12 rounds of injection and selection, RNAs enrichment of the pool with RNAs containing NLEs. In of both pools localized in the nucleus as efficiently as did contrast, the distribution of several non-selected control U2 snRNA. snRNAs remained constant throughout the selection pro- cedure (Figure 1B). Similarities in transport and maturation of Sm One known NLE is the Sm site of snRNAs to which nuclear localized RNAs (NL-RNAs) Sm proteins bind, promoting snRNP import (Fischer Individual cDNAs, made from nuclear localized Sm NL- et al., 1993). Therefore, if this selection process works as RNAs were cloned and sequenced (Figure 2A). All of the 794 Nuclear localization of stable RNAs Fig. 2. Selected Sm NL-RNAs. (A) Sequences of Sm NL-RNAs after 12 rounds of selection–amplification. The randomized region (N20), nucleotide changes and deletions (Δ) within the fixed sequence of the carrier RNA (dashed lines) are indicated; the 3 ends of the primers used for reverse transcription and PCR amplification are shown by arrows. Sequences of Sm protein binding sites are shown in bold letters. NL-101 RNA (top line) was used for testing the functionality of the selected Sm sites (see text). (B) Transport and maturation of NL-101 RNA. 1–2 fmol each of 32 7 P-labeled, m G-capped NL-101 and U5 RNAs were co-injected into nuclei (lanes 2–7) or cytoplasms (lanes 8–11) of oocytes. Oocytes were fractionated into nuclei (N) and cytoplasms (C) at 2 h (lanes 2 and 5), 3 h (lanes 8 and 10), 5 h (lanes 3 and 6) or 24 h (lanes 4, 7, 9 and 11) after injection. Total RNAs of each fraction were analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) and autoradiography of the gel. M, RNAs prior to injection. Cap hypermethylation and RNP formation was assayed 24 h after nuclear injection (cf. lane 4) by immunoprecipitation of 7 2,2,7 total nuclear RNAs (T, lane 12) or nuclear extracts using antibodies specific for the mono- (m G; lanes 13 and 14) or hypermethylated (m G; lanes 15 and 16) cap structures, or for Sm proteins (lanes 17 and 18), respectively. RNAs in the precipitate (P) and supernatant (S) fractions were analyzed by denaturing PAGE. Sm RNAs contained a consensus sequence (AAU- one or two nucleotides (compare lanes 7 and 4, or lanes UUUUGG) which resembled a typical Sm site; ~10% of 9 and 11), as was observed also with U1 RNA (Yang the cloned RNAs were Sm contaminants that did not et al., 1992) and U5 RNA. When injected into the localize in nuclei, when tested individually (data not cytoplasm (lanes 8–11), similar 3 end shortenings shown). Interestingly, this consensus sequence is always occurred prior to (lane 9) and after (lane 11) nuclear located close to the U1 3 stem–loop of the carrier RNA. import. Immunoprecipitations (lanes 13–18) of RNAs or This preference for position is in agreement with the RNPs present in the nucleus at 24 h after nuclear injection finding that the function of an Sm site in nucleo–cyto- (lane 4 and lane 12) demonstrated that all of the NL-101 plasmic trafficking is dependent on the presence and nature RNA was associated with Sm proteins (lanes 17 and 18) 2,2,7 of adjacent stem–loop structures (Jarmolowski and Mattaj, and had acquired a hypermethylated m G-cap structure 1993). The occasional deletions and nucleotide changes (lanes 13–16). Therefore, transport, 3 end trimming, Sm outside the randomized region are probably caused by the protein binding and cap hypermethylation of NL-101 RNA amplification method, since they occur at positions close closely resembled comparable steps in the maturation of to the endpoints of the primers; whether they are important snRNAs and their precursors. These results show that the parts of the selected sequences of Sm NL-RNAs is method used here is capable of selecting RNA molecules unclear. on the basis of their abilities to be transported within cells. The individual Sm NL-RNAs follow the transport and maturation pathway of spliceosomal snRNAs like U1 Three groups of secondary structures common to 7 – and U5. When injected into nuclei of oocytes, m G-capped Sm NL-RNAs NL-101 RNA was exported to the cytoplasm where its 3 In contrast to Sm NL-RNAs, no strongly conserved end was shortened (Figure 2B, lanes 2–7). After import sequence motif was evident in the N20 region of the back into the nucleus, the 3 end was further trimmed by selected Sm NL-RNAs, other than a bias against 795 C.Grimm, E.Lund and J.E.Dahlberg Fig. 3. Selected Sm NL-RNAs. (A) Nucleotide sequence and predicted RNA structures of Sm NL-RNAs. Representation of the sequences is as in Figure 2A. Asterisks indicate sequences with potential Sm sites (not tested). NL-RNAs, indicated by bold face were used further in this study. Missing dashes indicate uncertainties in the sequence. Brackets indicate groups of RNA sequences with similar secondary structures, as predicted using the RNA fold method of Zuker (1989). Circled nucleotides in the RNA structures show the sequences selected from the random library. Gray dots indicate the 5 m G-cap structures. (B) Structure probing of NL-15 RNA. 5 end- labeled (cap-labeled, see Materials and methods) NL-15 RNA was digested with single-strand specific RNase One (One) or double-strand specific RNase V1 (V1) for 2 min (lanes 4 and 8), 6 min (lanes 5 and 9) or 18 min (lanes 6 and 10). Digestion products were analyzed by denaturing PAGE. Control incubations (C; lanes 3 and 7) were done in buffer for 18 min; RNase A (lane 1) and RNase T1 (lane 2) partial digests were used for RNA sequencing. The RNase V1 cleavage products, which contain 3 OH groups migrate ~1 nucleotide slower than products of comparable length generated by the other RNases containing 3 P ends. Open symbols, cleavage by RNase One; filled symbols, cleavage by RNase V1; triangles and circles, strongly and weakly cleaved sites, respectively; arrow, A17 (see text). adenosine residues. As with the Sm NL-RNAs, almost 3A). All of the proposed structures contained strong stems all of the Sm NL-RNAs had several nucleotides deleted at (group I and II) or near (group III) the 5 end and most 3 of the N20 region (Figure 3A). However, a variant of nucleotides derived from the randomized sequence (circled NL-15 RNA containing the complete sequence of the in Figure 3A) were in strongly base-paired regions. The carrier RNA localized efficiently in the nuclei of oocytes possible structures of four RNAs (NL-4, -5, -8 and -17; demonstrating that the nuclear localization of the selected bottom of Figure 3A) did not fit any of the three RNA was not dependent on the deletion of these nucleo- categories; three of these RNAs (NL-4, -5 and -17) were tides (data not shown). tested individually and showed only inefficient nuclear Using the RNA M-fold method (Zuker, 1989), we localization (data not shown); they were not tested further. – – categorized the selected Sm NL-molecules into three The proposed structures of the Sm NL-RNAs are groups according to possible secondary structures (Figure supported by the digestion pattern of NL-15 RNA (a 796 Nuclear localization of stable RNAs Fig. 4. Nuclear localization and stability of wild-type and mutant NL-25 RNAs. Wild-type (top panel) and mutant m G-capped NL-25 RNAs were injected into nuclei (lanes 2–7) or cytoplasms (lanes 8–11) of oocytes. Oocytes were fractionated and RNAs analyzed as in Figure 2B. The RNA secondary structures were predicted as in Figure 3A. Brackets in NL-25 mark the sequences that are altered in the mutant RNAs. NL-25/mut1: 5-proximal half of the stem mutated; NL-25/mut2: 5-distal half of the stem mutated; NL-25/mut12: both halves mutated (compensatory mutations). M, RNAs prior to injection; N, nuclear RNAs; C, cytoplasmic RNAs. member of group I) produced by both the single-strand RNA. We note that unlike NL-25/mut12 RNA, all of specific RNase One and the double-strand specific RNase the selected Sm NL-RNAs contain imperfect 5 stem V1 (Figure 3B). Cleavage at A17 by RNase V1 might structures (cf. Figure 3A and data not shown), which indicate stacking of A17 between the two stems on might be important for RNA–protein interactions needed either side. for stabilization and nuclear localization of the RNAs (see The RNAs shown in Figure 3A had been selected by Discussion). Finally, NL-25/mut1 RNA does not localize 11 rounds of nuclear injection, followed by a 12th round in the nucleus even though it contains the sequence of cytoplasmic injection to select for RNAs that also can selected from the random library, showing that the selected be imported from the cytoplasm. However, a comparison primary sequence alone is not sufficient for retention of these RNAs to RNAs that had not been subjected to this and import; instead, the sequence probably is important last selection step did not reveal any obvious differences in because it contributes to the formation of a specific RNA type of sequence (low content of adenosine residues), structure. RNA structure or transport behavior (data not shown). The transport behaviors of individual members of the three structural groups supported the importance of the 5 Importance of the 5 stem for nuclear localization stem for nuclear localization of Sm NL-RNAs (Figure of Sm NL-RNAs 5A). Whereas most molecules of the original RNA pool To determine whether the structures of Sm NL-RNAs localized in the cytoplasm (top panel), the in vivo selected are important for nuclear localization, we disrupted the 5 Sm RNAs of group I and group II were retained in stem of NL-25 RNA by mutagenesis of the DNA template. the nucleus and were imported when injected into the Sequence alterations in either one side (NL-25/mut1) or cytoplasm (second and third panels). NL-39 RNA (group the other side (NL-25/mut2) of the stem led to similar III) however, apparently reached an equilibrium between decreases in RNA stability and caused cytoplasmic nucleus and cytoplasm 24 h after injection (last panel, accumulation of the mutant RNAs (Figure 4, second and compare lanes 4 and 7). We note that the 5 end of NL- third panels). In contrast, the RNA with compensatory 39 RNA is only weakly base-paired (G–U pairing), in mutations that resulted in reformation of a strong 5 stem contrast to the 5 ends of RNAs in groups I and II (NL-25/mut12; Figure 4, last panel) again was localized (Figure 3A). in the nucleus. However, this latter RNA was less stable The 5 end of NL-15 RNA appeared to be masked and less efficiently imported than the original NL-25 since neither the intracellular localization nor the stability 797 C.Grimm, E.Lund and J.E.Dahlberg – 7 Fig. 5. Nuclear localization of individual Sm NL-RNAs and sequestration of the m G-cap as an export signal. (A) Nucleo–cytoplasmic distribution 7 – of m G-capped RNAs from the starting pool (round 1 of the selection) and from the three structural groups of selected Sm NL-RNAs (round 12, see Figure 3A). Oocyte injection, fractionation and RNA analysis was as in Figure 2B. (B and C) Role of the m G-cap as an export signal. (B) Nucleo–cytoplasmic distributions of m G- and ApppG-capped NL-15 RNA with a 5 extension (NL-15/5Ext) were assayed 2 h after nuclear 7 – injection and compared with those of m G-capped wild-type NL-15 and U1 Sm RNAs. M, RNAs prior to injection; N, nuclear RNAs; C, cytoplasmic RNAs. (C) Comparison of nuclear localizations of capped (m G-) and uncapped (pppG-) NL-15 RNAs. RNAs were analyzed as in (A). of the RNA was affected by the presence or absence of a NL-15/5Ext RNA was exported as efficiently as U1 5-cap (Figure 5C). Also, the capped RNA was poorly snRNA (panels 2 and 4); in contrast, the A-capped RNA precipitated by cap-specific antibodies (data not shown). remained in the nucleus (panel 3). These results indicate To test if the proximity of the 5-cap to the body of the that localization of wild-type NL-15 RNA in the nucleus 7 7 structured RNA interfered with recognition of the m G- is due, at least in part, to the inability of the 5 m G-cap cap as an export signal, we extended the 5 end of the to interact with CBC. This conclusion is supported by our RNA with a short unstructured sequence (Figure 5B, finding that the efficiency of UV-crosslinking between a NL-15/5Ext). Cap-specific antibodies could now access component of CBC and the 5 m G-cap of NL-15 RNA the cap and efficiently precipitate the RNA (data not is strongly enhanced when the RNA has the 5 extension shown). Since the extension should make the cap access- (see Figure 8A, below). ible to other proteins as well, it should allow interaction between the m G-cap and proteins of the CBC (Izaurralde Complexes between NL-15 RNA and nuclear et al., 1995) and consequently promote export. As a proteins control, we injected NL-15/5Ext RNA bearing an ApppG To test whether NL-15 RNA is retained in the nucleus as (A-cap) which is a poor substrate for CBC binding. Unlike a consequence of its binding to a nuclear protein, high 7 7 m G-capped wild-type NL-15 RNA (panel 1), m G-capped amounts of unlabeled competitor RNAs were injected to 798 Nuclear localization of stable RNAs Fig. 6. Saturation of nuclear retention of NL-15 RNA. 1 to 2 fmol of 32 7 – P-labeled m G-capped NL-15, U1 Sm and U3 RNAs were co- injected into nuclei of Xenopus oocytes in the absence (lanes 1–9) or presence of 500 fmol of unlabeled m G-capped NL-15 (lanes 10–18) or U1 (lanes 19–27) competitor RNAs. Nucleo–cytoplasmic distribution was tested 1 h (lanes 2, 6, 11, 15, 20 and 24), 2 h (lanes 3, 7, 12, 16, 21 and 25), 4 h (lanes 4, 8, 13, 17, 22 and 26) and 24 h (lanes 5, 9, 14, 18, 23 and 27) after injection as in Figure 2B. M, RNAs prior to injection; N, nuclear RNAs; C, cytoplasmic RNAs. saturate a potential nuclear retention site(s) (Figure 6). Injection of 500 fmol of NL-15 competitor RNA caused destabilization and cytoplasmic accumulation of labeled NL-15 RNA, but it had no effect on retention of U3 RNA. Likewise, injection of 500 fmol of U1 RNA did not affect nuclear retention of NL-15 RNA but it did saturate export of U1Sm RNA (a mutant form of U1 RNA that is exported but cannot be re-imported into nuclei, Mattaj and de Robertis, 1985). Thus, nuclear retention of NL-15 RNA involves a specific and saturable factor(s) that is not Fig. 7. Complex formation of NL-15 RNA with La protein. required for U3 retention or U1 export. (A) Formation of complexes in nuclear extracts from Xenopus oocytes. To learn which nuclear proteins might interact with 10 fmol of m G-capped NL-15 RNA was incubated either in buffer NL-15 RNA, we incubated the RNA in nuclear extracts (lane 1) or in 0.5 oocyte equivalents of nuclear extract in the absence and assayed for RNA–protein complex formation by native (lane 2) or presence of 2.5 ng (lanes 3, 7 and 11), 10 ng (lanes 4, 8 and 12), 50 ng (lanes 5, 9 and 13) or 200 ng (lanes 6, 10 and 14) of gel electrophoresis (Figure 7). The complex that formed unlabeled competitor RNA and subsequently analyzed by native between NL-15 RNA and a component(s) of the nuclear PAGE. λ competitor: 172 nucleotides long RNA made from λ DNA. extract (Figure 7A, lane 2) was specific, since it could be (B) Formation of complexes in immunodepleted extracts. 10 fmol of competed efficiently by an excess of unlabeled NL-15 m G-capped NL-15 RNA was incubated either in buffer (lane 1), in 0.5 oocyte equivalents of untreated nuclear extract (lane 2) or in RNA (lanes 3–6), but only poorly by an unrelated RNA extract immunodepleted with anti-La antibodies (lane 3) or total (lanes 11–14). U6 RNA also competed for complex human IgGs (lane 4). (C) Supershifts of NL-15 complexes with anti- formation (lanes 7–10); since U6 RNA binds La protein La antibodies. Formation of complexes of m G-capped NL-15 in in nuclei of Xenopus oocytes (Terns et al., 1992), we nuclear extract from Xenopus oocytes (left panel) or extract from tested whether the complex formed with NL-15 RNA E.coli (right panel) as in (A). RNA was incubated either in buffer (lanes 1 and 7), in nuclear extract of Xenopus oocytes (lanes 2–6) or involved La protein. A variety of experiments shows that in extract of E.coli cells that have (i; lanes 8 and 9) or have not been this is the case (Figure 7B–D): (i) NL-15 RNA did induced (ni; lane 10) to express recombinant human La protein. not form a complex in nuclear extracts that had been Complexes formed in nuclear extracts from Xenopus oocytes were immunologically depleted of La protein (Figure 7B). incubated further with increasing amounts of anti-La antibodies (B- (ii) The complex that formed in untreated extracts was 103; lanes 3–6). The complex formed in extracts from E.coli that contained human La protein was incubated with the amount of anti-La supershifted by the addition of anti-La antibodies (Figure antibodies (B-103) used in lane 6 (lane 9). F, free RNA; C, complex; 7C, lanes 1–6); the supershift could be reversed by the S, supershift. (D) Acceleration of hY1 RNA export by high levels of addition of recombinant human La protein (data not 32 NL-15 RNA. P-labeled hY1 RNA was injected into nuclei of shown). (iii) NL-15 RNA formed a similar complex in Xenopus oocytes in the absence (–; top panel) or presence (; bottom panel) of 500 fmol of unlabeled NL-15 RNA. Oocytes were extracts made from Escherichia coli cells that expressed fractionated and RNAs analyzed as in Figure 2B. recombinant human La protein, but not in control E.coli extracts; this complex also was supershifted by anti-La antibodies (Figure 7C, lanes 7–10). (iv) NL-15 RNA that was injected in nuclei of Xenopus oocytes was acceleration of export of hY1 RNA occurs when hY1 coprecipitated with anti-La antibodies (data not shown). RNA is prevented from binding to La protein by mutation (iv) NL-15 RNA competed for a U6 complex formed (Simons et al., 1996), the effect of NL-15 RNA is probably in nuclear extracts (data not shown). (vi) High levels of due to competition of the two RNAs for available La NL-15 RNA dramatically accelerated export of hY1 RNA protein. We conclude that NL-15 RNA binds La protein from the nucleus in vivo (Figure 7D); because a comparable in nuclei (and cytoplasms; see below) of Xenopus oocytes. 799 C.Grimm, E.Lund and J.E.Dahlberg Fig. 9. Import of NL-15 RNA via a protein pathway. Inhibition of import of NL-15 RNA by WGA. The RNA mixture shown in lane 1 (M) was injected into cytoplasms of oocytes that had () or had not (–) been pre-injected with WGA. Nucleo–cytoplasmic distribution of the RNAs were analyzed 20 h after RNA injection as in Figure 2B. protein (lane 1), but depletion of the extract by anti-CBP 20 antibodies resulted in loss of labeling of the smallest protein (lane 2). Similarly, immunodepletion of La protein Fig. 8. Crosslinking of proteins to NL-15 RNA by UV light. from the extracts (lane 3) greatly reduced the labeling of (A) Crosslinking to RNA injected into nuclei of Xenopus oocytes. 7 the 49 kDa protein, as did the addition of uncapped m G-cap labeled NL-15 (lanes 1 and 2) or NL-15/5Ext (lane 3) RNA competitor NL-15 RNA (lane 4). We conclude that the was injected into nuclei of oocytes. 15–30 min after injection 10 nuclei were isolated, pooled, homogenized and spun for 3 min at smallest and largest proteins are CBP 20 and La protein, 8000 g. The cleared supernatant was irradiated on ice for respectively. The identity of the ~22 kDa protein has not 45 min, treated with RNase A and T1 and fractionated on a 12% been determined. SDS–PAGE. (B) Crosslinking to RNA incubated in nuclear extract. The precise location in NL-15 RNA to which La binds m G-cap labeled NL-15/5Ext RNA was incubated in nuclear extract of four oocyte-equivalents. UV-crosslinking, RNase treatment and has not been determined. However, the 3 half of NL-15 SDS–PAGE as in (A). Molecular size markers are indicated on the can be deleted without affecting binding of La to the RNA left. (data not shown), suggesting an internal sequence and/or structure as the La binding site; not a 3 uridylate stretch However, we cannot exclude the possibility that additional like that to which La binds in U6 RNA. The striking proteins also bind NL-15 RNA. increase in label transfer to CBP 20 upon removal of La protein (Figure 8A, lane 2) indicates that bound La protein Crosslinking of NL-15 RNA to nuclear proteins inhibits access of CBC to the 5-cap of NL-15 RNA, either The binding of NL-15 RNA to La protein was confirmed sterically or through stabilization of an RNA structure that by transfer of label from the 5-cap of NL-15 RNA to masks the cap. Thus, the appearance of NL-15 RNA in proteins in nuclei of oocytes upon UV-crosslinking. Sev- the cytoplasm after injection of high levels of the RNA eral polypeptides were labeled (Figure 8A, lane 1), the into the nucleus (Figure 6) may result in part from most highly labeled of which (indicated by * ) had the unmasking of the 5 cap rather than from the saturation mobility of La protein (49 kDa). Labeling of this of a nuclear retention site. protein was greatly reduced by the co-injection of uncapped NL-15 competitor RNA (lane 2). Surprisingly, Proteins involved in nuclear import of selected the presence of the uncapped competitor RNA also resulted NL-RNAs in an increased labeling of the fastest migrating protein In the final round of the selection procedures RNAs (lane 2). Three proteins (including a third protein with an were injected into the cytoplasm but re-isolated from the apparent molecular weight of ~22 kDa) were labeled very nucleus, thereby imposing a requirement that the selected efficiently when NL-15 RNA carrying the 5 extension RNAs have the capacity to be imported into the nucleus. (NL-15/5Ext, see Figure 5B) was injected (lane 3). These It is likely that the selected Sm RNAs are imported by results indicate that the 49 kDa protein is probably La the same mechanisms as those used normally for Sm protein, which binds to the NL-15 RNA regardless of its spliceosomal RNPs since these two classes of RNAs cap status. However, it binds close enough to the cap to undergo similar maturation events in the cytoplasm, be cross-linked to it and to reduce the interaction of the such as binding Sm proteins and cap hypermethylation cap with the other proteins. The 5 extension of NL-15/ (Figure 2B). In support of this proposal, import of the 5Ext RNA allows the smaller proteins to interact with selected Sm RNA was hardly affected by the lectin the cap, even in the presence of the 49 kDa protein. WGA (data not shown), an effective inhibitor of import of Because of its size and the fact that uncapped competitor NLS-containing proteins but not of spliceosomal snRNPs RNA did not reduce its labeling (Figure 8B), the smallest (Fischer et al., 1991). protein is very likely to be the small subunit of CBC. In contrast, uptake of NL-15 RNA, like that of U6 The identities of the proteins were confirmed in vitro RNA, was strongly inhibited by injection of WGA under by immunodepletion of GV extracts prior to RNA addition conditions where import of U5 RNA was unaffected and UV-crosslinking (Figure 8B). When incubated in (Figure 9). This sensitivity to the lectin indicates that untreated extracts, NL-15/5Ext RNA labeled all three NL-15 RNA, like U6 RNA, probably is imported through 800 Nuclear localization of stable RNAs Fig. 10. Protein requirements for nuclear import of NL-15 RNA. (A) Blockage of formation of RNA–La complexes by anti-La antibodies. 32 7 7 P-labeled U5 (m G-capped), U6 (γ-mpppG-capped) and NL-15 (m G-capped) RNAs were co-injected into cytoplasms of oocytes that had been pre-injected with IgGs from normal human serum (lanes 1–3) or GO anti-La antibodies (α-La; lanes 4–6). Complex formation between La protein and RNAs was tested 9 h after RNA injection by immunoprecipitations of cytoplasmic extracts with B-103 anti-La antibodies. RNAs were prepared from total extract (T), precipitate (P) and supernatant (S) and analyzed by PAGE. (B) Blockage of nuclear import of NL-15 RNA by anti-La antibodies. RNAs were injected into oocytes that had been pre-injected either with IgGs (lanes 2 and 3) or GO anti-La antibodies (α-La; lanes 4 and 5) as in (A). Nucleo–cytoplasmic distribution was assayed 9 h after injection as in Figure 2B. M, RNAs prior to injection; C, cytoplasmic RNAs; N, nuclear RNAs. Quantitation of the RNAs in lanes 2–5 was done by PhosphorImager analyses and import in the presence of IgGs (empty bars) or anti-La antibodies (hatched bars) was expressed as [N/(NC)]100%. (C) Different requirements for nuclear import of NL-15 and U6 RNA. 1 to 32 7 7 2 fmol each of P-labeled NL-15 (m G-capped), U6 (γ-mpppG-capped) and U1 (m G-capped) RNAs were co-injected into cytoplasms of Xenopus A– oocytes in the absence (lanes 1–4) or presence of 500 fmol unlabeled NL-15 (lanes 5–8) or U6 (lanes 9–12) competitor RNAs. Nucleo–cytoplasmic distributions were assayed 3 h (lanes 1, 3, 5, 7, 9 and 11) and 24 h (lanes 2, 4, 6, 8, 10 and 12) after injection as in Figure 2B. C, cytoplasmic RNAs; N, nuclear RNAs. its binding to an NLS-containing protein in an energy of the other RNA (Figure 10C) also demonstrates that requiring process. This is supported by our finding that migration of the two RNAs into the nucleus is mediated import of NL-15 RNA does not occur when the oocytes by different factors. are incubated at 4°C (data not shown). If La is involved in import of NL-15 RNA, one might Since La protein can bind to NL-15 RNA in the nucleus, expect that high levels of La binding RNAs such as U6 we tested whether the La protein present in the cytoplasm and hY1 would inhibit NL-15 import. However, the 3 (Peek et al., 1993) might bind NL-15 RNA and influence ends of both U6 and hY1 RNAs are removed when they its nuclear import. After cytoplasmic injection NL-15, and are injected at high levels into the cytoplasm of oocytes to a lesser extent U6 RNA, could be coprecipitated with (Figure 10C, lane 10 and data not shown), thereby losing anti-La antibodies from the cytoplasm (Figure 10A, lanes their La binding sites as suggested also by Simons et al. 2 and 3). Preinjection of anti-La (but not control IgG) (1996). Consistent with this, we found that injection of as antibodies inhibited the formation of complexes between much as 2 pmol of hY1 RNA in the cytoplasm failed to NL-15 or U6 RNAs and La protein (lanes 4 and 5). The fully prevent complex formation between La protein and anti-La antibodies reduced the fraction of NL-15 RNA NL-15 RNA (data not shown). Therefore, although both that was imported into the nucleus by ~4-fold (Figure U6 and hY1 RNAs destabilize NL-15 RNA to some extent 10B, lanes 3 and 5 and right hand panel), but had no (Figure 10C, lane 10 and data not shown), they have only effect on import of U6 and U5 snRNAs. This suggests a minor effect on NL-15 RNA import. that import of NL-15 RNA is dependent specifically on We propose that NL-15 RNA is imported as a con- complex formation with cytoplasmic La protein, whereas sequence of its ability to bind to cytoplasmic La protein. import of U6 (and U5) is not. The failure of high levels In that sense, the RNA might use La as an NLS presenting of NL-15 or U6 RNA to compete for nuclear import carrier protein (or as a promoter of interaction with a 801 C.Grimm, E.Lund and J.E.Dahlberg carrier protein) for its nuclear import. In contrast, import consensus found here would affect the ability of U1 RNA of U6 RNA which is not dependent on interaction with to function in pre-mRNA splicing. La, probably requires another, yet unidentified protein. Structures of Sm RNAs selected for nuclear localization Discussion In contrast to the Sm NL-RNAs, no consensus nucleotide sequence motif could be found in the selected Sm NL- The work presented here shows that Xenopus oocytes can RNAs. However, the 5 regions of all of these RNAs be used to select RNAs that localize to specific sub-cellular could be folded into structures that contained extended compartments. The selected NLEs had to overcome the stems, with several bulged nucleotides and/or small loops inherent rapid export characteristics of the m G-capped not conserved in sequence or position (Figure 3 and data RNA carrier derived from pre-U1RNA, either by adding not shown). Disruption of the proposed structure by signals that would direct import back from the cytoplasm, substitution of blocks of nucleotides in NL-25 RNA by inactivating the export signals or by adding nuclear reduced its nuclear localization and stability, and compens- retention signals. Two motifs were isolated, one of which atory substitutions designed to re-establish a double- was known from previous work, and the other of which stranded structure restored these features significantly, but is novel. Undoubtedly, other motifs could be isolated in not completely (Figure 4). Because restoration of the similar selections, since known NLEs, such as the box D nuclear localization and stability of the doubly mutant sequences of nucleolar snoRNAs, were not found in RNA was incomplete, we suggest that the unpaired this screen. nucleotides in the stem region of NL-25 RNA are important for the interaction of this RNA with cellular proteins In vivo selection of functional Sm protein binding and for nuclear localization. We note that the selected sequences sequences are particularly low in adenosine residues Since Sm protein binding promotes nuclear import of (Figure 3A) and we speculate that this bias in sequence RNAs, we expected that one class of the RNAs selected was caused by conversion of adenosines to inosines in for nuclear localization would contain Sm protein binding double-stranded stems (Polson and Bass, 1994) during sites. Such a class was enriched by precipitation with anti- in vivo selection. Sm antibodies. All of the selected molecules contained The selected NLEs of Sm NL-RNAs may function in the motif AAUUUUUGG, located near the 3 stem of nuclear localization by masking features in the carrier the carrier RNA (Figure 2A). This consensus sequence RNA that would otherwise promote export. In particular, strongly resembles other Sm protein binding sites both in the 5 m G-caps of NL-RNAs are located adjacent to the sequence and in location, near an RNA stem–loop structure RNA duplexes which may prevent their recognition as an (Jarmolowski and Mattaj, 1993). The ability of the selected export signal. Although the m G-cap structure is not Sm sites in the NL-RNAs to function both in transport essential for export of pre-snRNAs, it increases the export and cap hypermethylation (Figure 2B) validated the use efficiency of the RNAs (Hamm and Mattaj, 1990; Terns of in vivo selection in the isolation of authentic RNA et al., 1993) by providing the binding site for the proteins localization elements. Furthermore the homogeneity of of CBC (Izaurralde et al., 1995). The importance of an the isolated A U G consensus makes it unlikely that accessible 5-cap for export of NL-15 RNA is demon- 2 5 2 completely unrelated sequences or structures can function strated by the appearance in the cytoplasm of a variant of efficiently in Sm protein binding. However, the possibility NL-15 RNA in which the 5-cap was at the end of a exists that other Sm binding sites were present in the single-stranded extended region. molecules precipitated after round 4, but that these other sites were unable to function effectively enough in either Nuclear retention of Sm NL-RNAs import or stabilization of the RNA to survive all rounds The appearance of NL-15 RNA in the cytoplasm also of selection. This may explain why most of the RNAs could be promoted by nuclear injection of large amounts that were coprecipitated with Sm proteins after four rounds of competitor NL-15 RNA (Figure 6), presumably as a of selection were only poorly imported in round 5 (Figure consequence of saturation of a limited number of specific 1B). We are currently sequencing some of these early retention sites in the nucleus. Several other nuclear RNAs, Sm NL-RNAs to test the existence of unrelated Sm such as U6 spliceosomal RNA and the snoRNAs U3 and protein binding sites. U8, appear to have specific nuclear retention signals Although the RNA carrier for the randomized sequences (Hamm and Mattaj, 1989; Terns et al., 1993, 1995; Terns was derived from U1 RNA, the Sm sites of the selected and Dahlberg, 1994; Boelens et al., 1995; our unpublished NL-RNAs differ from those of most U1 RNAs, in which results); the differential saturation of nuclear retention for the U stretch is interrupted by single nucleotide changes U3, U8 and U6 RNAs indicates the available number of (AAUUUGUGG in human, rat, mouse, chicken and bean special retention factors(s) each RNA is limited for (Terns U1 RNAs; AAUUUCUGG in frog U1 RNAs; Reddy and et al., 1995; our unpublished results). However, such Busch, 1988, and references therein). We note that the experiments cannot distinguish between retention of an Sm NL-RNAs were not selected for their ability to RNA as a result of binding to a factor that anchors it to function in RNA processing. As was shown previously, a nuclear structure versus binding to a molecule that close agreement with the consensus Sm binding site, masks an export signal. while increasing the efficiency of nuclear localization and One nuclear factor that may contribute to the nuclear stability, may interfere with the function of certain Sm localization of NL-15 RNA, in part by masking the m G- RNAs (Grimm et al., 1993). It is unclear whether the cap export signal, is La protein. NL-15 RNA associates 802 Nuclear localization of stable RNAs with this abundant, predominantly nuclear protein, as hence leads to their inability to compete for La binding. assayed by gel shift experiments, UV-crosslinking and Previously, Simons and co-workers (1994) reported that immunoprecipitations with anti-La antibodies. Moreover, La protein dissociates from hY1 RNA during or after nuclear injection of high levels of NL-15 RNA accelerates nuclear export and suggested that this is caused by a 3 the export of wild-type hY1 RNA, an RNA that binds La end processing event which removes the La binding site protein in the nucleus and normally is exported very (Simons et al., 1996). Our findings are in agreement with slowly (Simons et al., 1994; Figure 7D). A mutation in this proposal. hY1 RNA which removes its La binding site causes the Since nuclear uptake of U6 RNA was unaffected by rapid export of the RNA, indicating that La binding is antibodies to La protein or high levels of NL-15 competitor responsible for nuclear retention of hY1 RNA (Simons RNA (Figure 10), we conclude that La protein is not et al., 1996). Accordingly, we propose that high levels of needed for U6 import. Similarly, high levels of poly(ACG), NL-15 RNA effectively reduce the nuclear pool of free a competent inhibitor for La binding (D.Kenan, personal La protein so that most of the hY1 RNA is not bound by communication), reduced the import of NL-15 RNA but La, and thus can be exported rapidly. We conclude that was without effect on U6 and U1 import (data not shown). NL-15 RNA binds La protein in vivo and that this Thus, NL-15 and U6 RNAs define two similar but separate interaction is involved in the retention of NL-15 in nuclei RNA import pathways, both of which differ from the of Xenopus oocytes. pathway used to import Sm snRNPs. La protein can bind either to uridylate-rich 3 ends (Stefano, 1984) or to internal sequences (Chang et al., In vivo selection of RNAs from combinatorial 1994; D.Kenan, personal communication) of RNAs. libraries Because NL-15 RNA contains no 3 uridylates, and The isolation of functional Sm sites with a strong consensus deletion of the 3 stem–loop of NL-15 RNA does not motif, and the identification of a novel RNA structural prevent its binding to La (data not shown), the site in this element in the selection for NLEs demonstrates the feasi- RNA that is recognized by La protein is probably within bility of using an in vivo selection method to isolate RNAs the 5 duplex structure. Proximity of the 5-cap and the with desired intracellular localization characteristics. The La protein binding site also is indicated by the UV- method identifies RNAs within a combinatorial library of mediated transfer of label from the cap to bound La molecules that are both stable in the cell and have the protein. By binding close to the cap of NL-15 RNA, La selected localization property. protein apparently interferes with the recognition of the Xenopus laevis oocytes are ideal cells for this type of 5-cap of the RNA by CBC, a nuclear export factor for selection since they can readily be microinjected and snRNAs (Figure 8A). This interference could be direct fractionated. Moreover, these cells have the capacity to through competition for binding to a site in the RNA or deal with large numbers of molecules, allowing for the indirect through stabilization of a structure that masks the use of reasonably large pools of RNAs in the first rounds 5-cap. The appearance of NL-15 RNA in the cytoplasm of in vivo selection. We are modifying this selection in the presence of high levels of NL-15 competitor RNA method to study other mechanisms that contribute to RNA (Figure 6) thus may reflect saturation of La protein and transport and intracellular localization. possibly other nuclear factors, leaving the 5-cap available The RNAs selected in this study show that several for interaction with CBC. mechanisms can be used, alone or in concert, to localize RNAs in cell nuclei. Likewise, an NLE, such as the La A novel role for La protein as a mediator of RNA protein binding site, may support nuclear localization in import from the cytoplasm to the nucleus more than one way. To survive the final round of selection, the NL-RNAs had to have the ability to be imported into the nucleus. Unlike Materials and methods the import of Sm NL-RNAs (which occurs via the snRNP pathway), import of NL-15 RNA was strongly inhibited DNA templates and in vitro transcription DNA templates for in vitro transcription were generated by PCR by the lectin WGA (Figure 9), indicating that the RNA is amplification of RNA coding regions using appropriate primer pairs. brought into the nucleus complexed with an NLS-con- Templates used to transcribe U1, U2, U3 and U6 RNAs were described taining protein, as is U6 RNA (Fischer et al., 1991). previously (Terns et al., 1993, 1995). The template for U5 RNA was Furthermore, both the formation of complexes between generated by PCR amplification of the X.laevis X.l.U5 11H gene (Kazmaier et al., 1987) using a 5 primer containing the SP6 promoter NL-15 RNA and La in the cytoplasm, and the import of sequence (5-GGAATTCGATTTAGGTGACACTATAGAATACTCTG- NL-15 RNA into the nucleus were inhibited by cytoplasmic GTTTCT-3) and a 3 primer with a two nucleotide extension to make injection of antibodies that recognized La protein. These precursor length U5 RNA (5-AGTACCTGGTGTGAACCAGGC-3). results suggest that the complex responsible for import of The template for hY1 RNA was described previously (Simons et al., NL-15 RNA either contains La or requires La for its 1994). In vitro transcription of T7 or SP6 DNA templates was done in 20 μl reactions containing 40 mM Tris–HCl pH 7.9, 6 mM MgCl , formation. 2 2 mM spermidine, 10 mM NaCl, 0.1 mg/ml BSA, 10 mM DTT, 2–4 The failure of other La binding RNAs such as U6 units RNasin, 0.3 mM rATP, rCTP and rUTP, 0.1 mM rGTP plus 20 μCi (Figure 10C) and hY1 RNA (data not shown) to compete 32 7 [α- P]rGTP (25 pmol) and either 0.5 mM m GpppG or ApppG-cap for nuclear import of NL-15 RNA most likely is due to dinucleotide (NEB) or 2 mM γ-mpppG (kindly provided by Ram Reddy); incubation was for 1–2 h at 37°C with 20 units of either T7 or SP6 their inability to interact efficiently with La protein in the RNA polymerase. Unlabeled competitor RNAs were prepared in 100 μl cytoplasm. Both U6 and hY1 RNA normally bind La reactions containing 80 mM HEPES–KOH [N-(2-hydroxyethyl)piper- protein through their 3 uridylate stretch, but both RNAs azine-N-(2-ethanesulfonic acid)] pH 7.5, 16 mM MgCl , 2 mM spermi- are trimmed in the cytoplasm when injected at high levels. dine, 40 mM DTT, 2 mM rATP, rCTP, rUTP, 0.2 mM rGTP and 1 mM This results in the loss of their La binding site and (m GpppG) or 2 mM (γ-mpppG) cap analog. Incubation was for 2 h 803 C.Grimm, E.Lund and J.E.Dahlberg with 100 units of RNA polymerase, followed by a second addition of NL-15 RNA. Cleavage with RNase V1 (Pharmacia) was done with 0.7 100 units of RNA polymerase and further incubation for 2 h. All units of enzyme at 22°C in a 100 μl reaction containing 10 mM Tris RNA transcripts were purified by electrophoresis in a 8% denaturing (pH 7.5), 10 mM MgCl , 50 mM KCl, 10 μg tRNA and 50 fmol of NL- polyacrylamide gel and elution in 0.3 M NaCl, 10 mM Tris–HCl pH 7.6, 15 RNA. After 2, 6 and 18 min, 25 μl aliquots were removed and the 0.1 mM EDTA and 0.5% SDS. reactions were stopped by the addition of SDS (to a final concentration of 0.1%) and 10 μg yeast RNA. RNAs were prepared immediately by Oocyte injection and analysis of RNA transport phenol–chloroform (24:1) extraction and ethanol precipitation. Cleavage –4 Nuclei or cytoplasms of intact stage V and VI oocytes from X.laevis with RNase A (ICN) and RNase T1 (Calbiochem) was done with 10 were injected with 12 nl of H O containing 1–10 fmol of P-labeled units or 1 unit of enzyme, respectively for 18 min at 55°C in 7 M urea, RNAs and where indicated, different amounts of unlabeled competitor 1 mM EDTA, 25 mM Na-Acetate pH 7.0 and 10 μg tRNA. For controls, RNAs. The injection mixture also contained blue dextran to monitor the NL-15 RNA was incubated in buffer without enzyme under the respective accuracy of nuclear injection (Jarmolowski et al., 1994). After incubation conditions for 18 min. RNase cleavage products were separated on a at 18°C for different times (see figure legends), oocytes were manually 10% polyacrylamide gel containing 8.3 M urea. dissected under mineral oil (Lund and Paine, 1990) into nuclear and cytoplasmic fractions. After proteinase K digestion, total RNAs were Site directed mutagenesis of individual RNAs isolated from each fraction by two extractions with phenol–chloroform NL-25 and NL-15 RNAs were mutagenized by PCR amplification (24:1) and ethanol precipitations and purified RNAs were analyzed by using the following sets of primers. For NL-25/mut1: 5 primer SP6-U1 electrophoresis in 8% polyacrylamide gels containing 7 M urea. 5mut (5-GAATTCGATTTAGGTGACACTATAGAATACTATGGTG- GCAGGGG-3) and 3 primer CS536; for NL-25/mut2: 5 primer T7 In vivo selection SELEX and 3 primer NL-25/mut2 (5-ATCAGGGGAAAGCGCG- The DNA template used to transcribe the pool of RNAs for the first AACGCAGTCCACTACCAGAATACTATGGAAAGTCCTCAGGG- round of selection was prepared by annealing 50 pmol of an 87 nucleotide 3); for NL-25/mut12: 5 primer SP6-U1 5mut and 3 primer NL-25/ oligonucleotide (complementary to the RNA sequence shown in Figure mut2; for NL-15/5Ext: 5 primer SP6-NL-RS (5-GGAATTCGATTTA- 1A) to 250 pmol of a partially overlapping oligonucleotide containing GGTGACACTATAGAACTAGAGTACTGGGATACTTACCTGGCA- the T7 promoter sequence plus nucleotides 1–19 of the RNA shown in GGGG-3) and 3 primer CS536. PCR products were purified by Figure 1A (T7 SELEX: 5-AATGTCGACTAATACGACTCACTATA- electrophoresis in a 6% polyacrylamide gel and used for in vitro GGGATACTTACCTGGCAGG-3). After annealing at 60°C, the products transcription. were extended with Stoffel enzyme (Perkin Elmer) for1hat 60°C. Full-length double-stranded products were purified by electrophoresis in Antibodies, immunoprecipitations and immunodepletions 2,2,7 a 6% polyacrylamide gel. For generation of the starting pool of RNAs, Rabbit polyclonal antibodies against the m G- (Bringmann et al., 250 ng of the gel-purified template was transcribed with T7 RNA 1983; kindly provided by R.Lu¨hrmann) and the m G-cap (Munns et al., polymerase and RNAs were purified as described above. For the first 1982; kindly provided by T.Munns) were used to precipitate deproteinized round of selection, 50 fmol of the experimental RNAs were injected RNAs, mouse monoclonal antibodies against Sm proteins (mAb Y12, together with 1–2 fmol each of the control RNAs into nuclei or Lerner et al., 1981; kindly provided by J. Steitz) and anti-La antibodies cytoplasms of 50 oocytes. Theoretically, this corresponded to 2.510 from human patient sera (B-103, GO; kindly provided by D.Kenan and molecules and thus could contain all of the 1.110 different molecules J.Keene) were used to precipitate RNPs from nuclear and cytoplasmic that can be formed from a 20 nucleotide long randomized sequence. extracts and deplete nuclear extracts of La protein. Anti-CBP20 antibodies After 20–24 h of incubation at 18°C, oocytes were dissected into nuclei (rabbit; Izaurralde et al., 1995; kindly provided by E.Izaurralde and and cytoplasms and total RNA prepared from both compartments. I.Mattaj) were used to immunodeplete nuclear extract of CBC. Analytical polyacrylamide gels were used to determine the nucleo– Immunoprecipitations were done as described previously (Terns et al., cytoplasmic distribution of experimental and control RNAs at each round 1992). For the injection of anti-La antibodies, total IgGs were purified of selection. Prior to reverse transcription and PCR amplification (RT– from serum GO, essentially as described (Harlow and Lane, 1988). PCR), the experimental RNA in the nuclear fraction was size selected IgGs from 1.2 ml serum were bound to protein A–Sepharose beads in a and purified by electrophoresis in a 8% polyacrylamide gel containing 1.5 ml column in 100 mM Tris–HCl (pH 8). The column was washed 7 M urea. Reverse transcription was done in a 20 μl reaction containing with 10 column volumes of 100 mM Tris–HCl (pH 8) followed by 10 50 mM Tris–HCl pH 8.5, 75 mM KCl, 10 mM DTT, 3 mM MgCl , column volumes of 10 mM Tris (pH 8) and IgGs were eluted with 0.5 mM dNTPs, 2–4 units RNasin and 5 μM primer CS536 (5- 100 mM glycine (pH 3). After the addition of 0.1 volumes of1MTris– ATCAGGGGAAAGCGCGAACGCAGTCC-3). The mixture was HCl (pH 8), the neutralized IgGs were precipitated by the addition heated for 2 min at 95°C and cooled to 37°C prior to the addition of 1 of 1 volume of saturated (NH ) SO and collected by 30 min of 4 2 4 μl of M-MLV reverse transcriptase (200 units/μl, USB). After incubation centrifugation at 3000 g. The pellet was drained, resuspended in 0.1 ml for 15 min at 37°C, 80 μl of a mixture containing 1.8 mM MgCl , 50 of PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na HPO , 1.8 mM KH PO , 2 2 4 2 4 mM KCl, 10 mM Tris–HCl pH 9.0, 0.1% Triton X-100, 8 μM primer pH 7.4) and dialyzed against 3 2000 ml of PBS. IgGs were further T7 SELEX and 0.5 units of Taq DNA polymerase (Promega) were concentrated ~8-fold using a microconcentrator (microcon 100; Amicon). added and overlayed with mineral oil to prevent condensation. PCR Anti-La activity of the concentrated IgGs was tested in separate gel shift amplification was done using 35 cycles of denaturation (1 min at 95°C), experiments (data not shown) and 60 nl (per oocyte) of the solution annealing (45 s at 68°C) and extension (1 min at 72°C). RT–PCR containing 3 mM DTT and 4 units RNasin/μl were injected into the products were fractionated by electrophoresis in a 6% polyacrylamide cytoplasm of each oocyte. gel and the purified DNA templates were used to transcribe RNA for Anti-CBP20 or anti-La (GO) antibodies were coupled to protein A– the next round of selection. The total amounts of RNA used for injection Sepharose beads and used to immunodeplete nuclear extract from La were: 50 (rounds 1–4) or 2–10 (rounds 5–12) fmol per oocyte and the protein or CBC, respectively. Extract from 50 nuclei was incubated with number of oocytes injected were 50 (rounds 1–2), 30 (round 3) or 5–10 the respective antibodies for 1.5 h on ice with occasional stirring. The (rounds 4–12). The RT–PCR products after the 12th round of selection mixes were spun for 10 s and the supernatants used as immunode- were re-amplified using a 5 primer containing a HindIII restriction site pleted extracts. and a 3 primer containing a EcoRI restriction site. These two sites were used for cloning of the PCR products into pGEM-4Z vector (Promega). Complex formation and native gel electrophoresis Escherichia coli cells were transfected by electroporation and plasmids Nuclear extracts from oocytes were prepared as described (Terns et al., were isolated from individual colonies. Inserts were sequenced by the 1995). For complex formation, 10 fmol (1 μl) of m G-capped NL-15 or dideoxy termination method using Sequenase version 2.0 (USB). γ-mpppG-capped U6 RNA were mixed with 50 ng of 23S rRNA (1 μl) and 8 μl (0.5 oocyte equivalents) of nuclear extract in D buffer Structure probing (250 mM sucrose, 25 mM KCl, 5 mM MgCl ,3 mM DTT,50mM The substrate for structure probing in solution was in vitro prepared Tris–HCl, pH 7.6). After incubation for 20 min at 19°C, 2.5 μlof NL-15 RNA with a single label in the m G-cap structure. Unlabeled loading solution (50% glycerol, 2.5 mM EDTA, 0.01% bromphenol- 7 32 NL-15 RNA was m G-capped using guanylyltransferase plus [α- P] blue, 0.01% xylene cyanol) was added and the samples were fractionated GTP and S-adenosylmethionine (SAM) as described (Terns et al., 1995). immediately in native 6% polyacrylamide gels (30:0.8) in 0.5 TEB Cleavage with RNase One (Promega) was done with 0.03 units of (1 TEB: 90 mM Tris, 90 mM boric acid, 2.5 mM EDTA). For enzyme at 22°C in a 100 μl reaction containing 10 mM Tris–HCl pH supershifts, the samples with preformed complexes (see above) were 7.5, 5 mM EDTA, 200 mM Na-Acetate, 10 μg tRNA and 50 fmol of incubated for 20 min on ice with 1 μl anti-La antibodies B-103 (diluted 804 Nuclear localization of stable RNAs in D and mixed with RNasin) prior to addition of the loading solution. Davis,L.I. (1995) The nuclear pore complex. Annu. Rev. Biochem., 64, The gels were run at room temperature for ~2 h at 10 V/cm, fixed in a 865–896. solution containing 10% acetic acid and 20% methanol for 25 min and Eckner,R., Ellmeier,W. and Birnstiel,M.L. (1991) Mature mRNA 3 end dried prior to autoradiography. formation stimulates RNA export from the nucleus. EMBO J., 10, 3513–3522. Escherichia coli extracts containing human La protein Finlay,D.R., Newmeyer,D.D., Price,T.M. and Forbes,D.J. (1987) Escherichia coli cells [strain BL21(DE3) pLysS] expressing recombinant Inhibition of in vitro nuclear transport by a lectin that binds to nuclear human La protein were kindly provided by D.Kenan. Cells were grown pores. J. Cell Biol., 104, 189–200. in LB in a 50 ml culture; expression of La protein was induced by Fischer,U., Darzynkiewicz,E., Tahara,S.M., Dathan,N.A., Lu¨hrmann,R. the addition of IPTG (isopropyl β-D-thiogalactopyranoside) to a final and Mattaj,I.W. (1991) Diversity in the signals required for nuclear concentration of 0.4 mM. 3 h after induction, PMSF (phenylmethyl- accumulation of U snRNPs and variety in the pathways of nuclear sulfonyl fluoride) was added to a final concentration of 0.125 mg/ml transport. J. Cell Biol., 113, 705–714. and cells were harvested by centrifugation at 5000 g for 10 min at Fischer,U., Sumpter,V., Sekine,M., Satoh,T. and Lu¨hrmann,R. (1993) room temperature. Cells were resuspended in 2 ml of 25 mM Tris–HCl Nucleo-cytoplasmic transport of U snRNPs: definition of a nuclear pH 8.0, 3 mM MgCl , 0.1 mM EDTA, 0.5 mM DTT, 100 mM NaCl, location signal in the Sm core domain that binds a transport receptor 0.125 mg/ml PMSF and quickly frozen in a dry-ice ethanol bath. Cells independently of the m G-cap. EMBO J., 12, 573–583. were thawed in a 37°C water bath in the presence of freshly added Fischer,U., Meyer,S., Teufel,M., Heckel,C., Lu¨hrmann,R. and PMSF and sonicated for 2 30 s (Branson sonifier, setting 2). After Rautmann,G. (1994) Evidence that HIV-1 Rev directly promotes the centrifugation at 12 000 g for 30 min at 4°C, the cleared supernatant nuclear export of unspliced RNA. EMBO J., 13, 4105–4112. was collected and fresh PMSF added. The extract was stored at 4°C and Fischer,U., Huber,J., Boelens,W.C., Mattaj,I.W. and Lu¨hrmann,R. (1995) used as source for human La protein. Extracts from uninduced cells The HIV-1 Rev activation domain is a nuclear export signal that were prepared as above, except that no IPTG was added. 1 μl extract accesses an export pathway used by specific cellular RNAs. Cell, 82, (diluted 1:16 in D ) was used for complex formation as described 475–483. above. Forbes,D.J. (1992) Structure and function of the nuclear pore complex. Annu. Rev. Cell Biol., 8, 495–527. UV-crosslinking Go¨rlich,D. and Mattaj,I.W, (1996) Nucleocytoplasmic transport. Science, 10–30 fmol of RNA, labeled only in its 5 m G-cap (Terns et al., 1995), 271, 1513–1518. were incubated in nuclear extracts as for complex formation (see above) Grimm,C., Stefanovic,B. and Schu¨mperli,D. (1993) The low abundance in the absence or presence of unlabeled competitor RNAs as indicated of U7 snRNA is partly determined by its Sm binding site. EMBO J., in the figure legends. The extracts were either untreated or depleted 12, 1229–1238. from La protein or CBC. After 20 min of incubation, the samples were Grimm,C., Lund,E. and Dahlberg,J.E. (1995) In vivo selection of RNA irradiated with UV light (short wavelength) for 45 min on ice. The sequences involved in nucleocytoplasmic RNA trafficking. Nucleic samples were then treated with RNase T1 and RNase A for 1 h at 37°C. Acids Symp. Ser., 33, 34–36. Proteins were concentrated by the addition of 5 volumes of acetone and Hamm,J. and Mattaj,I.W. (1989) An abundant U6 snRNP found in germ centrifugation. 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(1994) Retention and 5 cap trimethylation of U3 snRNA in the nucleus. Science, 264, 959–961. Terns,M.P., Lund,E. and Dahlberg,J.E. (1992) 3 end-dependent formation of U6 small nuclear ribonucleoprotein particles in Xenopus laevis oocyte nuclei. Mol. Cell. Biol., 12, 3032–3040. Terns,M.P., Dahlberg,J.E. and Lund,E. (1993) Multiple cis-acting signals for export of pre-U1 snRNA from the nucleus. Genes Dev., 7, 1898–1908. Terns,M.P., Grimm,C., Lund,E. and Dahlberg,J.E. (1995) A common maturation pathway for small nucleolar RNAs. EMBO J., 14, 4860– Tuerk,C. and Gold,L. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science, 249, 505–510. Vankan,P., McGuigan,C. and Mattaj,I.W. (1990) Domains of U4 and U6 snRNAs required for snRNP assembly and splicing complementation in Xenopus oocytes. EMBO J., 9, 3397–3404. Yang,H., Moss,M.L., Lund,E. and Dahlberg,J.E. (1992) Nuclear processing of the 3 terminal nucleotides of pre-U1 RNA in Xenopus laevis oocytes. Mol. Cell. Biol., 12, 1553–1560. Zapp,M.L. (1992) RNA nucleocytoplasmic transport. Semin. Cell Biol., 3, 289–297. Zuker,M. (1989) On finding all suboptimal foldings of an RNA molecule. Science, 244, 48–52. Received on December 14, 1995; revised on September 30, 1996 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

In vivo selection of RNAs that localize in the nucleus

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Copyright © European Molecular Biology Organization 1997
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0261-4189
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10.1093/emboj/16.4.793
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Abstract

The EMBO Journal Vol.16 No.4 pp.793–806, 1997 1992; Izaurralde and Mattaj, 1992, 1995). Other nuclear Christian Grimm , Elsebet Lund and RNAs, like the small nucleolar RNAs (snoRNAs) and U6 James E.Dahlberg snRNA are not exported to the cytoplasm, but are retained Department of Biomolecular Chemistry, 1300 University Avenue, in the nucleus (Vankan et al., 1990; Terns and Dahlberg, University of Wisconsin, Madison, WI 53706, USA 1994; Boelens et al., 1995; Terns et al., 1995). Present address: Department of Ophthalmology, University Hospital- Several pathways exist for nuclear import of RNA Zu¨rich, 8091 Zu¨rich, Switzerland (Fischer et al., 1991; Michaud and Goldfarb, 1992). Corresponding author Efficient import of most spliceosomal snRNAs into nuclei, as snRNPs, requires two cis-acting elements: a binding Nuclear localization of an RNA is affected by cis- site for the Sm proteins and a trimethylguanosine cap acting elements (NLEs) that lead to nuclear import or structure (reviewed in Nigg et al., 1991; Izaurralde and retention or to blockage of export from the nucleus. Mattaj, 1992). In contrast, U6 snRNA (Hamm and Mattaj, To identify such elements, we selected and analyzed 1989) and 5S rRNA (Allison et al., 1993) lack both of transcripts that localized in the nuclei of Xenopus laevis these signals, but nevertheless can be imported into nuclei oocytes. The RNAs were isolated from a collection of of Xenopus oocytes, bound to proteins that contain nuclear m G-capped RNAs in which a combinatorial library localization signals (NLSs) (Fischer et al., 1991). (n  20) of sequences had been inserted. One class of Likewise, several distinct pathways are likely to exist selected RNAs (Sm ) had a consensus Sm binding also for RNA export, as demonstrated by the lack of site (AAUUUUUGG) and bound Sm proteins in the competition in this process between different classes cytoplasm; these RNAs resembled small nuclear RNAs of RNA molecules (Jarmolowski et al., 1994; Pokrywka like U1 and U5 RNAs in their bi-directional nucleo– and Goldfarb, 1995) and by the differential inhibition of cytoplasmic transport and their 5-cap hyper- export by various inhibitors of NPC function (E.Lund methylation. Another class, Sm RNAs, contained and J.E.Dahlberg, in preparation; Powers et al., 1997). sequences that masked the m G-caps of the RNAs However, several RNAs also share aspects of common and promoted interaction with La protein. These export pathways. For example, saturation of the Rev- RNAs were retained within nuclei after nuclear injec- mediated export of RRE-containing RNAs (Fischer et al., tion and were imported when injected into the cyto- 1994) affects export of pre-snRNAs and 5S RNA, but not plasm. Their nuclear import and retention were that of mRNAs and tRNAs (Fischer et al., 1995). independent of a 5-cap, required an imperfect double- The monomethylated cap structure of pre-snRNAs and stranded stem near the 5 end, and depended on mRNAs facilitates export of these RNAs to the cytoplasm interaction with La protein. Import of the Sm RNAs, (Hamm and Mattaj, 1990; Terns et al., 1993; Jarmolowski while using the import pathway of proteins, was distinct et al., 1994). This m G-cap is recognized by the proteins from that of U6 RNA. of the cap binding complex (CBC) which mediates export, Keywords: La protein/nuclear localization elements at least of snRNAs (Izaurralde et al., 1995; reviewed in (NLEs)/RNA transport/selection of RNAs in vivo/Sm Go¨rlich and Mattaj, 1996). Other structural elements of consensus site these RNAs are also important for export (Hamm and Mattaj, 1990; Eckner et al., 1991; Terns et al., 1993; Jarmolowski et al., 1994). Several RNA binding proteins have been implicated in the export of different classes of Introduction RNAs, but only CBP20 and CBP80 (the proteins of CBC; Izaurralde et al., 1995), and the Rev protein (Fischer et al., The distribution of RNAs within various sub-cellular 1995), have been shown to promote export of bound RNA compartments results from a balance of export, import (reviewed in Izaurralde and Mattaj, 1995; Go¨rlich and and retention. In several instances, cis-acting sequences Mattaj, 1996). in the RNA and trans-acting cellular factors have been The lectin wheat germ agglutinin (WGA), which binds shown to contribute to these processes. However, relatively to N-acetylglucosamine residues of NPC proteins, is an few of the actual proteins and RNA signals are known. effective inhibitor of many types of nucleo–cytoplasmic Intracellular RNA transport is an active process that often transport (reviewed by Forbes, 1992). Import of most involves translocation of the RNAs (or RNPs) across the proteins carrying an NLS is inhibited by WGA treatment nuclear envelope, through the nuclear pore complexes (NPCs; Davis, 1995). Whereas most RNAs (mRNA, tRNA (Finlay et al., 1987; Dabauvalle et al., 1988) as is the and scRNA) are transported unidirectionally from the import of U6 RNA, which apparently occurs by the same nucleus to the cytoplasm, the precursors of many small pathway (Fischer et al., 1991; this study). In contrast, nuclear RNAs (pre-snRNAs) and 5S ribosomal RNA are import of snRNPs containing Sm proteins is relatively translocated through the pores in both directions (Zapp, insensitive to the lectin. Export of most RNAs is inhibited © Oxford University Press 793 C.Grimm, E.Lund and J.E.Dahlberg by WGA (E.Lund and J.E.Dahlberg, manuscript in pre- paration), but some of these effects may be secondary to inhibition of import of proteins that are required as carriers. Several mechanisms could account for the localization of RNAs in nuclei. RNAs may contain sequences that lead to their retention within the nucleus or to their import from the cytoplasm, as has been demonstrated for various classes of snRNAs. Examples include a sequence element common to most snoRNAs (box D), that is essential for nuclear retention of U8 snoRNA (Terns et al., 1995), and the binding site for Sm proteins of the spliceosomal RNAs U1, U2, U4 and U5, which promotes import of these molecules into the nucleus. Alternatively, structures or sequences that ordinarily would direct export to the cytoplasm may either be absent or masked in other RNAs that localize in nuclei. Here we define the term nuclear localization element (NLE) as any cis-acting RNA sequence or structural feature that promotes localization of the RNA in the nucleus. In this study we developed and used an iterative selection to identify signals and mechanisms that contribute to the localization of RNAs within nuclei. At least two classes of molecules were isolated that had the capacity to be localized within nuclei. One class of RNAs contains a consensus sequence for the Sm binding site (AAU- UUUUGG) that promotes nuclear localization of snRNAs; isolation of this expected class of molecules validated the method. Another class contains a structural motif that participates in nuclear localization of Sm RNAs. Inter- action of this structure with La protein appears to promote Fig. 1. Selection of RNAs with nuclear localization signals. retention of the RNA in the nucleus by masking the 5- (A) Structure of the RNA used for the selection of NLEs. Fixed cap, which otherwise acts as an export signal; furthermore, nucleotide sequences from the 5 and 3 ends of U1 snRNA are in by binding to La protein in the cytoplasm the RNA can upper case letters, and nucleotides in lower case letters indicate be imported in an NLS-dependent fashion. changes in the U1 sequences that were made to ensure efficient in vitro transcription by T7 RNA polymerase (addition of Gs at the 5 end) and increased in vivo stability of the RNA (A to C change in Results the 5 stem). N20 represents the 20 nucleotide randomized sequence. Arrows indicate the endpoints of the primers used for reverse In vivo selection of RNA elements that promote transcription and PCR amplification of the RNAs. (B) Enrichment of nuclear localization the pools for RNAs that localize in the nucleus. The percentages of RNAs that localized in the nucleus at 20–24 h after nuclear injection To select for NLEs (both nuclear import and nuclear were determined by PhosphorImager analysis of gels run after each retention signals), we used a short derivative of U1 round of selection and were expressed as [N/(NC)]100. Thick snRNA as the carrier molecule for a 20 nucleotide long, – lines, experimental RNA pools; thin lines, control U1Sm , U2 and U3 randomized sequence (N20; Figure 1A; see also Grimm RNAs that were exported to the cytoplasm, exported and imported back into the nucleus and retained in the nucleus, respectively. et al., 1995). Since the carrier moiety of this in vitro Sm and Sm pools of RNAs were generated at rounds 4 and 8 transcribed RNA contains a strong nuclear export signal (circles) by immunoprecipitation of nuclear extracts with anti-Sm in the form of the m G-cap (Hamm and Mattaj, 1990; antibodies; the RNAs in the precipitate (Sm ) and supernatant (Sm ) Izaurralde et al., 1995) but no nuclear import signal, most fractions were injected separately after round 4. of the stable molecules of the starting pool localized in the cytoplasm within 20 h after they had been injected expected, one class of the molecules we isolate should into nuclei (Figure 1B; see also Figure 5A, top panel). contain Sm protein binding sites. To distinguish between However, some RNAs received a sequence through the molecules that contain such Sm binding sites and mole- N20 insert that caused nuclear localization of the RNA, cules with other NLEs, we used anti-Sm antibodies to and therefore could be isolated from nuclei and amplified precipitate RNAs bound to Sm proteins (at rounds 4 and through multiple rounds of selection. The percentage of 8, as indicated by circles, Figure 1B). The resulting RNAs in the experimental RNA pool that localized in the Sm and Sm RNA pools were treated separately after nucleus increased with each round, indicating a gradual round 4. After 12 rounds of injection and selection, RNAs enrichment of the pool with RNAs containing NLEs. In of both pools localized in the nucleus as efficiently as did contrast, the distribution of several non-selected control U2 snRNA. snRNAs remained constant throughout the selection pro- cedure (Figure 1B). Similarities in transport and maturation of Sm One known NLE is the Sm site of snRNAs to which nuclear localized RNAs (NL-RNAs) Sm proteins bind, promoting snRNP import (Fischer Individual cDNAs, made from nuclear localized Sm NL- et al., 1993). Therefore, if this selection process works as RNAs were cloned and sequenced (Figure 2A). All of the 794 Nuclear localization of stable RNAs Fig. 2. Selected Sm NL-RNAs. (A) Sequences of Sm NL-RNAs after 12 rounds of selection–amplification. The randomized region (N20), nucleotide changes and deletions (Δ) within the fixed sequence of the carrier RNA (dashed lines) are indicated; the 3 ends of the primers used for reverse transcription and PCR amplification are shown by arrows. Sequences of Sm protein binding sites are shown in bold letters. NL-101 RNA (top line) was used for testing the functionality of the selected Sm sites (see text). (B) Transport and maturation of NL-101 RNA. 1–2 fmol each of 32 7 P-labeled, m G-capped NL-101 and U5 RNAs were co-injected into nuclei (lanes 2–7) or cytoplasms (lanes 8–11) of oocytes. Oocytes were fractionated into nuclei (N) and cytoplasms (C) at 2 h (lanes 2 and 5), 3 h (lanes 8 and 10), 5 h (lanes 3 and 6) or 24 h (lanes 4, 7, 9 and 11) after injection. Total RNAs of each fraction were analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) and autoradiography of the gel. M, RNAs prior to injection. Cap hypermethylation and RNP formation was assayed 24 h after nuclear injection (cf. lane 4) by immunoprecipitation of 7 2,2,7 total nuclear RNAs (T, lane 12) or nuclear extracts using antibodies specific for the mono- (m G; lanes 13 and 14) or hypermethylated (m G; lanes 15 and 16) cap structures, or for Sm proteins (lanes 17 and 18), respectively. RNAs in the precipitate (P) and supernatant (S) fractions were analyzed by denaturing PAGE. Sm RNAs contained a consensus sequence (AAU- one or two nucleotides (compare lanes 7 and 4, or lanes UUUUGG) which resembled a typical Sm site; ~10% of 9 and 11), as was observed also with U1 RNA (Yang the cloned RNAs were Sm contaminants that did not et al., 1992) and U5 RNA. When injected into the localize in nuclei, when tested individually (data not cytoplasm (lanes 8–11), similar 3 end shortenings shown). Interestingly, this consensus sequence is always occurred prior to (lane 9) and after (lane 11) nuclear located close to the U1 3 stem–loop of the carrier RNA. import. Immunoprecipitations (lanes 13–18) of RNAs or This preference for position is in agreement with the RNPs present in the nucleus at 24 h after nuclear injection finding that the function of an Sm site in nucleo–cyto- (lane 4 and lane 12) demonstrated that all of the NL-101 plasmic trafficking is dependent on the presence and nature RNA was associated with Sm proteins (lanes 17 and 18) 2,2,7 of adjacent stem–loop structures (Jarmolowski and Mattaj, and had acquired a hypermethylated m G-cap structure 1993). The occasional deletions and nucleotide changes (lanes 13–16). Therefore, transport, 3 end trimming, Sm outside the randomized region are probably caused by the protein binding and cap hypermethylation of NL-101 RNA amplification method, since they occur at positions close closely resembled comparable steps in the maturation of to the endpoints of the primers; whether they are important snRNAs and their precursors. These results show that the parts of the selected sequences of Sm NL-RNAs is method used here is capable of selecting RNA molecules unclear. on the basis of their abilities to be transported within cells. The individual Sm NL-RNAs follow the transport and maturation pathway of spliceosomal snRNAs like U1 Three groups of secondary structures common to 7 – and U5. When injected into nuclei of oocytes, m G-capped Sm NL-RNAs NL-101 RNA was exported to the cytoplasm where its 3 In contrast to Sm NL-RNAs, no strongly conserved end was shortened (Figure 2B, lanes 2–7). After import sequence motif was evident in the N20 region of the back into the nucleus, the 3 end was further trimmed by selected Sm NL-RNAs, other than a bias against 795 C.Grimm, E.Lund and J.E.Dahlberg Fig. 3. Selected Sm NL-RNAs. (A) Nucleotide sequence and predicted RNA structures of Sm NL-RNAs. Representation of the sequences is as in Figure 2A. Asterisks indicate sequences with potential Sm sites (not tested). NL-RNAs, indicated by bold face were used further in this study. Missing dashes indicate uncertainties in the sequence. Brackets indicate groups of RNA sequences with similar secondary structures, as predicted using the RNA fold method of Zuker (1989). Circled nucleotides in the RNA structures show the sequences selected from the random library. Gray dots indicate the 5 m G-cap structures. (B) Structure probing of NL-15 RNA. 5 end- labeled (cap-labeled, see Materials and methods) NL-15 RNA was digested with single-strand specific RNase One (One) or double-strand specific RNase V1 (V1) for 2 min (lanes 4 and 8), 6 min (lanes 5 and 9) or 18 min (lanes 6 and 10). Digestion products were analyzed by denaturing PAGE. Control incubations (C; lanes 3 and 7) were done in buffer for 18 min; RNase A (lane 1) and RNase T1 (lane 2) partial digests were used for RNA sequencing. The RNase V1 cleavage products, which contain 3 OH groups migrate ~1 nucleotide slower than products of comparable length generated by the other RNases containing 3 P ends. Open symbols, cleavage by RNase One; filled symbols, cleavage by RNase V1; triangles and circles, strongly and weakly cleaved sites, respectively; arrow, A17 (see text). adenosine residues. As with the Sm NL-RNAs, almost 3A). All of the proposed structures contained strong stems all of the Sm NL-RNAs had several nucleotides deleted at (group I and II) or near (group III) the 5 end and most 3 of the N20 region (Figure 3A). However, a variant of nucleotides derived from the randomized sequence (circled NL-15 RNA containing the complete sequence of the in Figure 3A) were in strongly base-paired regions. The carrier RNA localized efficiently in the nuclei of oocytes possible structures of four RNAs (NL-4, -5, -8 and -17; demonstrating that the nuclear localization of the selected bottom of Figure 3A) did not fit any of the three RNA was not dependent on the deletion of these nucleo- categories; three of these RNAs (NL-4, -5 and -17) were tides (data not shown). tested individually and showed only inefficient nuclear Using the RNA M-fold method (Zuker, 1989), we localization (data not shown); they were not tested further. – – categorized the selected Sm NL-molecules into three The proposed structures of the Sm NL-RNAs are groups according to possible secondary structures (Figure supported by the digestion pattern of NL-15 RNA (a 796 Nuclear localization of stable RNAs Fig. 4. Nuclear localization and stability of wild-type and mutant NL-25 RNAs. Wild-type (top panel) and mutant m G-capped NL-25 RNAs were injected into nuclei (lanes 2–7) or cytoplasms (lanes 8–11) of oocytes. Oocytes were fractionated and RNAs analyzed as in Figure 2B. The RNA secondary structures were predicted as in Figure 3A. Brackets in NL-25 mark the sequences that are altered in the mutant RNAs. NL-25/mut1: 5-proximal half of the stem mutated; NL-25/mut2: 5-distal half of the stem mutated; NL-25/mut12: both halves mutated (compensatory mutations). M, RNAs prior to injection; N, nuclear RNAs; C, cytoplasmic RNAs. member of group I) produced by both the single-strand RNA. We note that unlike NL-25/mut12 RNA, all of specific RNase One and the double-strand specific RNase the selected Sm NL-RNAs contain imperfect 5 stem V1 (Figure 3B). Cleavage at A17 by RNase V1 might structures (cf. Figure 3A and data not shown), which indicate stacking of A17 between the two stems on might be important for RNA–protein interactions needed either side. for stabilization and nuclear localization of the RNAs (see The RNAs shown in Figure 3A had been selected by Discussion). Finally, NL-25/mut1 RNA does not localize 11 rounds of nuclear injection, followed by a 12th round in the nucleus even though it contains the sequence of cytoplasmic injection to select for RNAs that also can selected from the random library, showing that the selected be imported from the cytoplasm. However, a comparison primary sequence alone is not sufficient for retention of these RNAs to RNAs that had not been subjected to this and import; instead, the sequence probably is important last selection step did not reveal any obvious differences in because it contributes to the formation of a specific RNA type of sequence (low content of adenosine residues), structure. RNA structure or transport behavior (data not shown). The transport behaviors of individual members of the three structural groups supported the importance of the 5 Importance of the 5 stem for nuclear localization stem for nuclear localization of Sm NL-RNAs (Figure of Sm NL-RNAs 5A). Whereas most molecules of the original RNA pool To determine whether the structures of Sm NL-RNAs localized in the cytoplasm (top panel), the in vivo selected are important for nuclear localization, we disrupted the 5 Sm RNAs of group I and group II were retained in stem of NL-25 RNA by mutagenesis of the DNA template. the nucleus and were imported when injected into the Sequence alterations in either one side (NL-25/mut1) or cytoplasm (second and third panels). NL-39 RNA (group the other side (NL-25/mut2) of the stem led to similar III) however, apparently reached an equilibrium between decreases in RNA stability and caused cytoplasmic nucleus and cytoplasm 24 h after injection (last panel, accumulation of the mutant RNAs (Figure 4, second and compare lanes 4 and 7). We note that the 5 end of NL- third panels). In contrast, the RNA with compensatory 39 RNA is only weakly base-paired (G–U pairing), in mutations that resulted in reformation of a strong 5 stem contrast to the 5 ends of RNAs in groups I and II (NL-25/mut12; Figure 4, last panel) again was localized (Figure 3A). in the nucleus. However, this latter RNA was less stable The 5 end of NL-15 RNA appeared to be masked and less efficiently imported than the original NL-25 since neither the intracellular localization nor the stability 797 C.Grimm, E.Lund and J.E.Dahlberg – 7 Fig. 5. Nuclear localization of individual Sm NL-RNAs and sequestration of the m G-cap as an export signal. (A) Nucleo–cytoplasmic distribution 7 – of m G-capped RNAs from the starting pool (round 1 of the selection) and from the three structural groups of selected Sm NL-RNAs (round 12, see Figure 3A). Oocyte injection, fractionation and RNA analysis was as in Figure 2B. (B and C) Role of the m G-cap as an export signal. (B) Nucleo–cytoplasmic distributions of m G- and ApppG-capped NL-15 RNA with a 5 extension (NL-15/5Ext) were assayed 2 h after nuclear 7 – injection and compared with those of m G-capped wild-type NL-15 and U1 Sm RNAs. M, RNAs prior to injection; N, nuclear RNAs; C, cytoplasmic RNAs. (C) Comparison of nuclear localizations of capped (m G-) and uncapped (pppG-) NL-15 RNAs. RNAs were analyzed as in (A). of the RNA was affected by the presence or absence of a NL-15/5Ext RNA was exported as efficiently as U1 5-cap (Figure 5C). Also, the capped RNA was poorly snRNA (panels 2 and 4); in contrast, the A-capped RNA precipitated by cap-specific antibodies (data not shown). remained in the nucleus (panel 3). These results indicate To test if the proximity of the 5-cap to the body of the that localization of wild-type NL-15 RNA in the nucleus 7 7 structured RNA interfered with recognition of the m G- is due, at least in part, to the inability of the 5 m G-cap cap as an export signal, we extended the 5 end of the to interact with CBC. This conclusion is supported by our RNA with a short unstructured sequence (Figure 5B, finding that the efficiency of UV-crosslinking between a NL-15/5Ext). Cap-specific antibodies could now access component of CBC and the 5 m G-cap of NL-15 RNA the cap and efficiently precipitate the RNA (data not is strongly enhanced when the RNA has the 5 extension shown). Since the extension should make the cap access- (see Figure 8A, below). ible to other proteins as well, it should allow interaction between the m G-cap and proteins of the CBC (Izaurralde Complexes between NL-15 RNA and nuclear et al., 1995) and consequently promote export. As a proteins control, we injected NL-15/5Ext RNA bearing an ApppG To test whether NL-15 RNA is retained in the nucleus as (A-cap) which is a poor substrate for CBC binding. Unlike a consequence of its binding to a nuclear protein, high 7 7 m G-capped wild-type NL-15 RNA (panel 1), m G-capped amounts of unlabeled competitor RNAs were injected to 798 Nuclear localization of stable RNAs Fig. 6. Saturation of nuclear retention of NL-15 RNA. 1 to 2 fmol of 32 7 – P-labeled m G-capped NL-15, U1 Sm and U3 RNAs were co- injected into nuclei of Xenopus oocytes in the absence (lanes 1–9) or presence of 500 fmol of unlabeled m G-capped NL-15 (lanes 10–18) or U1 (lanes 19–27) competitor RNAs. Nucleo–cytoplasmic distribution was tested 1 h (lanes 2, 6, 11, 15, 20 and 24), 2 h (lanes 3, 7, 12, 16, 21 and 25), 4 h (lanes 4, 8, 13, 17, 22 and 26) and 24 h (lanes 5, 9, 14, 18, 23 and 27) after injection as in Figure 2B. M, RNAs prior to injection; N, nuclear RNAs; C, cytoplasmic RNAs. saturate a potential nuclear retention site(s) (Figure 6). Injection of 500 fmol of NL-15 competitor RNA caused destabilization and cytoplasmic accumulation of labeled NL-15 RNA, but it had no effect on retention of U3 RNA. Likewise, injection of 500 fmol of U1 RNA did not affect nuclear retention of NL-15 RNA but it did saturate export of U1Sm RNA (a mutant form of U1 RNA that is exported but cannot be re-imported into nuclei, Mattaj and de Robertis, 1985). Thus, nuclear retention of NL-15 RNA involves a specific and saturable factor(s) that is not Fig. 7. Complex formation of NL-15 RNA with La protein. required for U3 retention or U1 export. (A) Formation of complexes in nuclear extracts from Xenopus oocytes. To learn which nuclear proteins might interact with 10 fmol of m G-capped NL-15 RNA was incubated either in buffer NL-15 RNA, we incubated the RNA in nuclear extracts (lane 1) or in 0.5 oocyte equivalents of nuclear extract in the absence and assayed for RNA–protein complex formation by native (lane 2) or presence of 2.5 ng (lanes 3, 7 and 11), 10 ng (lanes 4, 8 and 12), 50 ng (lanes 5, 9 and 13) or 200 ng (lanes 6, 10 and 14) of gel electrophoresis (Figure 7). The complex that formed unlabeled competitor RNA and subsequently analyzed by native between NL-15 RNA and a component(s) of the nuclear PAGE. λ competitor: 172 nucleotides long RNA made from λ DNA. extract (Figure 7A, lane 2) was specific, since it could be (B) Formation of complexes in immunodepleted extracts. 10 fmol of competed efficiently by an excess of unlabeled NL-15 m G-capped NL-15 RNA was incubated either in buffer (lane 1), in 0.5 oocyte equivalents of untreated nuclear extract (lane 2) or in RNA (lanes 3–6), but only poorly by an unrelated RNA extract immunodepleted with anti-La antibodies (lane 3) or total (lanes 11–14). U6 RNA also competed for complex human IgGs (lane 4). (C) Supershifts of NL-15 complexes with anti- formation (lanes 7–10); since U6 RNA binds La protein La antibodies. Formation of complexes of m G-capped NL-15 in in nuclei of Xenopus oocytes (Terns et al., 1992), we nuclear extract from Xenopus oocytes (left panel) or extract from tested whether the complex formed with NL-15 RNA E.coli (right panel) as in (A). RNA was incubated either in buffer (lanes 1 and 7), in nuclear extract of Xenopus oocytes (lanes 2–6) or involved La protein. A variety of experiments shows that in extract of E.coli cells that have (i; lanes 8 and 9) or have not been this is the case (Figure 7B–D): (i) NL-15 RNA did induced (ni; lane 10) to express recombinant human La protein. not form a complex in nuclear extracts that had been Complexes formed in nuclear extracts from Xenopus oocytes were immunologically depleted of La protein (Figure 7B). incubated further with increasing amounts of anti-La antibodies (B- (ii) The complex that formed in untreated extracts was 103; lanes 3–6). The complex formed in extracts from E.coli that contained human La protein was incubated with the amount of anti-La supershifted by the addition of anti-La antibodies (Figure antibodies (B-103) used in lane 6 (lane 9). F, free RNA; C, complex; 7C, lanes 1–6); the supershift could be reversed by the S, supershift. (D) Acceleration of hY1 RNA export by high levels of addition of recombinant human La protein (data not 32 NL-15 RNA. P-labeled hY1 RNA was injected into nuclei of shown). (iii) NL-15 RNA formed a similar complex in Xenopus oocytes in the absence (–; top panel) or presence (; bottom panel) of 500 fmol of unlabeled NL-15 RNA. Oocytes were extracts made from Escherichia coli cells that expressed fractionated and RNAs analyzed as in Figure 2B. recombinant human La protein, but not in control E.coli extracts; this complex also was supershifted by anti-La antibodies (Figure 7C, lanes 7–10). (iv) NL-15 RNA that was injected in nuclei of Xenopus oocytes was acceleration of export of hY1 RNA occurs when hY1 coprecipitated with anti-La antibodies (data not shown). RNA is prevented from binding to La protein by mutation (iv) NL-15 RNA competed for a U6 complex formed (Simons et al., 1996), the effect of NL-15 RNA is probably in nuclear extracts (data not shown). (vi) High levels of due to competition of the two RNAs for available La NL-15 RNA dramatically accelerated export of hY1 RNA protein. We conclude that NL-15 RNA binds La protein from the nucleus in vivo (Figure 7D); because a comparable in nuclei (and cytoplasms; see below) of Xenopus oocytes. 799 C.Grimm, E.Lund and J.E.Dahlberg Fig. 9. Import of NL-15 RNA via a protein pathway. Inhibition of import of NL-15 RNA by WGA. The RNA mixture shown in lane 1 (M) was injected into cytoplasms of oocytes that had () or had not (–) been pre-injected with WGA. Nucleo–cytoplasmic distribution of the RNAs were analyzed 20 h after RNA injection as in Figure 2B. protein (lane 1), but depletion of the extract by anti-CBP 20 antibodies resulted in loss of labeling of the smallest protein (lane 2). Similarly, immunodepletion of La protein Fig. 8. Crosslinking of proteins to NL-15 RNA by UV light. from the extracts (lane 3) greatly reduced the labeling of (A) Crosslinking to RNA injected into nuclei of Xenopus oocytes. 7 the 49 kDa protein, as did the addition of uncapped m G-cap labeled NL-15 (lanes 1 and 2) or NL-15/5Ext (lane 3) RNA competitor NL-15 RNA (lane 4). We conclude that the was injected into nuclei of oocytes. 15–30 min after injection 10 nuclei were isolated, pooled, homogenized and spun for 3 min at smallest and largest proteins are CBP 20 and La protein, 8000 g. The cleared supernatant was irradiated on ice for respectively. The identity of the ~22 kDa protein has not 45 min, treated with RNase A and T1 and fractionated on a 12% been determined. SDS–PAGE. (B) Crosslinking to RNA incubated in nuclear extract. The precise location in NL-15 RNA to which La binds m G-cap labeled NL-15/5Ext RNA was incubated in nuclear extract of four oocyte-equivalents. UV-crosslinking, RNase treatment and has not been determined. However, the 3 half of NL-15 SDS–PAGE as in (A). Molecular size markers are indicated on the can be deleted without affecting binding of La to the RNA left. (data not shown), suggesting an internal sequence and/or structure as the La binding site; not a 3 uridylate stretch However, we cannot exclude the possibility that additional like that to which La binds in U6 RNA. The striking proteins also bind NL-15 RNA. increase in label transfer to CBP 20 upon removal of La protein (Figure 8A, lane 2) indicates that bound La protein Crosslinking of NL-15 RNA to nuclear proteins inhibits access of CBC to the 5-cap of NL-15 RNA, either The binding of NL-15 RNA to La protein was confirmed sterically or through stabilization of an RNA structure that by transfer of label from the 5-cap of NL-15 RNA to masks the cap. Thus, the appearance of NL-15 RNA in proteins in nuclei of oocytes upon UV-crosslinking. Sev- the cytoplasm after injection of high levels of the RNA eral polypeptides were labeled (Figure 8A, lane 1), the into the nucleus (Figure 6) may result in part from most highly labeled of which (indicated by * ) had the unmasking of the 5 cap rather than from the saturation mobility of La protein (49 kDa). Labeling of this of a nuclear retention site. protein was greatly reduced by the co-injection of uncapped NL-15 competitor RNA (lane 2). Surprisingly, Proteins involved in nuclear import of selected the presence of the uncapped competitor RNA also resulted NL-RNAs in an increased labeling of the fastest migrating protein In the final round of the selection procedures RNAs (lane 2). Three proteins (including a third protein with an were injected into the cytoplasm but re-isolated from the apparent molecular weight of ~22 kDa) were labeled very nucleus, thereby imposing a requirement that the selected efficiently when NL-15 RNA carrying the 5 extension RNAs have the capacity to be imported into the nucleus. (NL-15/5Ext, see Figure 5B) was injected (lane 3). These It is likely that the selected Sm RNAs are imported by results indicate that the 49 kDa protein is probably La the same mechanisms as those used normally for Sm protein, which binds to the NL-15 RNA regardless of its spliceosomal RNPs since these two classes of RNAs cap status. However, it binds close enough to the cap to undergo similar maturation events in the cytoplasm, be cross-linked to it and to reduce the interaction of the such as binding Sm proteins and cap hypermethylation cap with the other proteins. The 5 extension of NL-15/ (Figure 2B). In support of this proposal, import of the 5Ext RNA allows the smaller proteins to interact with selected Sm RNA was hardly affected by the lectin the cap, even in the presence of the 49 kDa protein. WGA (data not shown), an effective inhibitor of import of Because of its size and the fact that uncapped competitor NLS-containing proteins but not of spliceosomal snRNPs RNA did not reduce its labeling (Figure 8B), the smallest (Fischer et al., 1991). protein is very likely to be the small subunit of CBC. In contrast, uptake of NL-15 RNA, like that of U6 The identities of the proteins were confirmed in vitro RNA, was strongly inhibited by injection of WGA under by immunodepletion of GV extracts prior to RNA addition conditions where import of U5 RNA was unaffected and UV-crosslinking (Figure 8B). When incubated in (Figure 9). This sensitivity to the lectin indicates that untreated extracts, NL-15/5Ext RNA labeled all three NL-15 RNA, like U6 RNA, probably is imported through 800 Nuclear localization of stable RNAs Fig. 10. Protein requirements for nuclear import of NL-15 RNA. (A) Blockage of formation of RNA–La complexes by anti-La antibodies. 32 7 7 P-labeled U5 (m G-capped), U6 (γ-mpppG-capped) and NL-15 (m G-capped) RNAs were co-injected into cytoplasms of oocytes that had been pre-injected with IgGs from normal human serum (lanes 1–3) or GO anti-La antibodies (α-La; lanes 4–6). Complex formation between La protein and RNAs was tested 9 h after RNA injection by immunoprecipitations of cytoplasmic extracts with B-103 anti-La antibodies. RNAs were prepared from total extract (T), precipitate (P) and supernatant (S) and analyzed by PAGE. (B) Blockage of nuclear import of NL-15 RNA by anti-La antibodies. RNAs were injected into oocytes that had been pre-injected either with IgGs (lanes 2 and 3) or GO anti-La antibodies (α-La; lanes 4 and 5) as in (A). Nucleo–cytoplasmic distribution was assayed 9 h after injection as in Figure 2B. M, RNAs prior to injection; C, cytoplasmic RNAs; N, nuclear RNAs. Quantitation of the RNAs in lanes 2–5 was done by PhosphorImager analyses and import in the presence of IgGs (empty bars) or anti-La antibodies (hatched bars) was expressed as [N/(NC)]100%. (C) Different requirements for nuclear import of NL-15 and U6 RNA. 1 to 32 7 7 2 fmol each of P-labeled NL-15 (m G-capped), U6 (γ-mpppG-capped) and U1 (m G-capped) RNAs were co-injected into cytoplasms of Xenopus A– oocytes in the absence (lanes 1–4) or presence of 500 fmol unlabeled NL-15 (lanes 5–8) or U6 (lanes 9–12) competitor RNAs. Nucleo–cytoplasmic distributions were assayed 3 h (lanes 1, 3, 5, 7, 9 and 11) and 24 h (lanes 2, 4, 6, 8, 10 and 12) after injection as in Figure 2B. C, cytoplasmic RNAs; N, nuclear RNAs. its binding to an NLS-containing protein in an energy of the other RNA (Figure 10C) also demonstrates that requiring process. This is supported by our finding that migration of the two RNAs into the nucleus is mediated import of NL-15 RNA does not occur when the oocytes by different factors. are incubated at 4°C (data not shown). If La is involved in import of NL-15 RNA, one might Since La protein can bind to NL-15 RNA in the nucleus, expect that high levels of La binding RNAs such as U6 we tested whether the La protein present in the cytoplasm and hY1 would inhibit NL-15 import. However, the 3 (Peek et al., 1993) might bind NL-15 RNA and influence ends of both U6 and hY1 RNAs are removed when they its nuclear import. After cytoplasmic injection NL-15, and are injected at high levels into the cytoplasm of oocytes to a lesser extent U6 RNA, could be coprecipitated with (Figure 10C, lane 10 and data not shown), thereby losing anti-La antibodies from the cytoplasm (Figure 10A, lanes their La binding sites as suggested also by Simons et al. 2 and 3). Preinjection of anti-La (but not control IgG) (1996). Consistent with this, we found that injection of as antibodies inhibited the formation of complexes between much as 2 pmol of hY1 RNA in the cytoplasm failed to NL-15 or U6 RNAs and La protein (lanes 4 and 5). The fully prevent complex formation between La protein and anti-La antibodies reduced the fraction of NL-15 RNA NL-15 RNA (data not shown). Therefore, although both that was imported into the nucleus by ~4-fold (Figure U6 and hY1 RNAs destabilize NL-15 RNA to some extent 10B, lanes 3 and 5 and right hand panel), but had no (Figure 10C, lane 10 and data not shown), they have only effect on import of U6 and U5 snRNAs. This suggests a minor effect on NL-15 RNA import. that import of NL-15 RNA is dependent specifically on We propose that NL-15 RNA is imported as a con- complex formation with cytoplasmic La protein, whereas sequence of its ability to bind to cytoplasmic La protein. import of U6 (and U5) is not. The failure of high levels In that sense, the RNA might use La as an NLS presenting of NL-15 or U6 RNA to compete for nuclear import carrier protein (or as a promoter of interaction with a 801 C.Grimm, E.Lund and J.E.Dahlberg carrier protein) for its nuclear import. In contrast, import consensus found here would affect the ability of U1 RNA of U6 RNA which is not dependent on interaction with to function in pre-mRNA splicing. La, probably requires another, yet unidentified protein. Structures of Sm RNAs selected for nuclear localization Discussion In contrast to the Sm NL-RNAs, no consensus nucleotide sequence motif could be found in the selected Sm NL- The work presented here shows that Xenopus oocytes can RNAs. However, the 5 regions of all of these RNAs be used to select RNAs that localize to specific sub-cellular could be folded into structures that contained extended compartments. The selected NLEs had to overcome the stems, with several bulged nucleotides and/or small loops inherent rapid export characteristics of the m G-capped not conserved in sequence or position (Figure 3 and data RNA carrier derived from pre-U1RNA, either by adding not shown). Disruption of the proposed structure by signals that would direct import back from the cytoplasm, substitution of blocks of nucleotides in NL-25 RNA by inactivating the export signals or by adding nuclear reduced its nuclear localization and stability, and compens- retention signals. Two motifs were isolated, one of which atory substitutions designed to re-establish a double- was known from previous work, and the other of which stranded structure restored these features significantly, but is novel. Undoubtedly, other motifs could be isolated in not completely (Figure 4). Because restoration of the similar selections, since known NLEs, such as the box D nuclear localization and stability of the doubly mutant sequences of nucleolar snoRNAs, were not found in RNA was incomplete, we suggest that the unpaired this screen. nucleotides in the stem region of NL-25 RNA are important for the interaction of this RNA with cellular proteins In vivo selection of functional Sm protein binding and for nuclear localization. We note that the selected sequences sequences are particularly low in adenosine residues Since Sm protein binding promotes nuclear import of (Figure 3A) and we speculate that this bias in sequence RNAs, we expected that one class of the RNAs selected was caused by conversion of adenosines to inosines in for nuclear localization would contain Sm protein binding double-stranded stems (Polson and Bass, 1994) during sites. Such a class was enriched by precipitation with anti- in vivo selection. Sm antibodies. All of the selected molecules contained The selected NLEs of Sm NL-RNAs may function in the motif AAUUUUUGG, located near the 3 stem of nuclear localization by masking features in the carrier the carrier RNA (Figure 2A). This consensus sequence RNA that would otherwise promote export. In particular, strongly resembles other Sm protein binding sites both in the 5 m G-caps of NL-RNAs are located adjacent to the sequence and in location, near an RNA stem–loop structure RNA duplexes which may prevent their recognition as an (Jarmolowski and Mattaj, 1993). The ability of the selected export signal. Although the m G-cap structure is not Sm sites in the NL-RNAs to function both in transport essential for export of pre-snRNAs, it increases the export and cap hypermethylation (Figure 2B) validated the use efficiency of the RNAs (Hamm and Mattaj, 1990; Terns of in vivo selection in the isolation of authentic RNA et al., 1993) by providing the binding site for the proteins localization elements. Furthermore the homogeneity of of CBC (Izaurralde et al., 1995). The importance of an the isolated A U G consensus makes it unlikely that accessible 5-cap for export of NL-15 RNA is demon- 2 5 2 completely unrelated sequences or structures can function strated by the appearance in the cytoplasm of a variant of efficiently in Sm protein binding. However, the possibility NL-15 RNA in which the 5-cap was at the end of a exists that other Sm binding sites were present in the single-stranded extended region. molecules precipitated after round 4, but that these other sites were unable to function effectively enough in either Nuclear retention of Sm NL-RNAs import or stabilization of the RNA to survive all rounds The appearance of NL-15 RNA in the cytoplasm also of selection. This may explain why most of the RNAs could be promoted by nuclear injection of large amounts that were coprecipitated with Sm proteins after four rounds of competitor NL-15 RNA (Figure 6), presumably as a of selection were only poorly imported in round 5 (Figure consequence of saturation of a limited number of specific 1B). We are currently sequencing some of these early retention sites in the nucleus. Several other nuclear RNAs, Sm NL-RNAs to test the existence of unrelated Sm such as U6 spliceosomal RNA and the snoRNAs U3 and protein binding sites. U8, appear to have specific nuclear retention signals Although the RNA carrier for the randomized sequences (Hamm and Mattaj, 1989; Terns et al., 1993, 1995; Terns was derived from U1 RNA, the Sm sites of the selected and Dahlberg, 1994; Boelens et al., 1995; our unpublished NL-RNAs differ from those of most U1 RNAs, in which results); the differential saturation of nuclear retention for the U stretch is interrupted by single nucleotide changes U3, U8 and U6 RNAs indicates the available number of (AAUUUGUGG in human, rat, mouse, chicken and bean special retention factors(s) each RNA is limited for (Terns U1 RNAs; AAUUUCUGG in frog U1 RNAs; Reddy and et al., 1995; our unpublished results). However, such Busch, 1988, and references therein). We note that the experiments cannot distinguish between retention of an Sm NL-RNAs were not selected for their ability to RNA as a result of binding to a factor that anchors it to function in RNA processing. As was shown previously, a nuclear structure versus binding to a molecule that close agreement with the consensus Sm binding site, masks an export signal. while increasing the efficiency of nuclear localization and One nuclear factor that may contribute to the nuclear stability, may interfere with the function of certain Sm localization of NL-15 RNA, in part by masking the m G- RNAs (Grimm et al., 1993). It is unclear whether the cap export signal, is La protein. NL-15 RNA associates 802 Nuclear localization of stable RNAs with this abundant, predominantly nuclear protein, as hence leads to their inability to compete for La binding. assayed by gel shift experiments, UV-crosslinking and Previously, Simons and co-workers (1994) reported that immunoprecipitations with anti-La antibodies. Moreover, La protein dissociates from hY1 RNA during or after nuclear injection of high levels of NL-15 RNA accelerates nuclear export and suggested that this is caused by a 3 the export of wild-type hY1 RNA, an RNA that binds La end processing event which removes the La binding site protein in the nucleus and normally is exported very (Simons et al., 1996). Our findings are in agreement with slowly (Simons et al., 1994; Figure 7D). A mutation in this proposal. hY1 RNA which removes its La binding site causes the Since nuclear uptake of U6 RNA was unaffected by rapid export of the RNA, indicating that La binding is antibodies to La protein or high levels of NL-15 competitor responsible for nuclear retention of hY1 RNA (Simons RNA (Figure 10), we conclude that La protein is not et al., 1996). Accordingly, we propose that high levels of needed for U6 import. Similarly, high levels of poly(ACG), NL-15 RNA effectively reduce the nuclear pool of free a competent inhibitor for La binding (D.Kenan, personal La protein so that most of the hY1 RNA is not bound by communication), reduced the import of NL-15 RNA but La, and thus can be exported rapidly. We conclude that was without effect on U6 and U1 import (data not shown). NL-15 RNA binds La protein in vivo and that this Thus, NL-15 and U6 RNAs define two similar but separate interaction is involved in the retention of NL-15 in nuclei RNA import pathways, both of which differ from the of Xenopus oocytes. pathway used to import Sm snRNPs. La protein can bind either to uridylate-rich 3 ends (Stefano, 1984) or to internal sequences (Chang et al., In vivo selection of RNAs from combinatorial 1994; D.Kenan, personal communication) of RNAs. libraries Because NL-15 RNA contains no 3 uridylates, and The isolation of functional Sm sites with a strong consensus deletion of the 3 stem–loop of NL-15 RNA does not motif, and the identification of a novel RNA structural prevent its binding to La (data not shown), the site in this element in the selection for NLEs demonstrates the feasi- RNA that is recognized by La protein is probably within bility of using an in vivo selection method to isolate RNAs the 5 duplex structure. Proximity of the 5-cap and the with desired intracellular localization characteristics. The La protein binding site also is indicated by the UV- method identifies RNAs within a combinatorial library of mediated transfer of label from the cap to bound La molecules that are both stable in the cell and have the protein. By binding close to the cap of NL-15 RNA, La selected localization property. protein apparently interferes with the recognition of the Xenopus laevis oocytes are ideal cells for this type of 5-cap of the RNA by CBC, a nuclear export factor for selection since they can readily be microinjected and snRNAs (Figure 8A). This interference could be direct fractionated. Moreover, these cells have the capacity to through competition for binding to a site in the RNA or deal with large numbers of molecules, allowing for the indirect through stabilization of a structure that masks the use of reasonably large pools of RNAs in the first rounds 5-cap. The appearance of NL-15 RNA in the cytoplasm of in vivo selection. We are modifying this selection in the presence of high levels of NL-15 competitor RNA method to study other mechanisms that contribute to RNA (Figure 6) thus may reflect saturation of La protein and transport and intracellular localization. possibly other nuclear factors, leaving the 5-cap available The RNAs selected in this study show that several for interaction with CBC. mechanisms can be used, alone or in concert, to localize RNAs in cell nuclei. Likewise, an NLE, such as the La A novel role for La protein as a mediator of RNA protein binding site, may support nuclear localization in import from the cytoplasm to the nucleus more than one way. To survive the final round of selection, the NL-RNAs had to have the ability to be imported into the nucleus. Unlike Materials and methods the import of Sm NL-RNAs (which occurs via the snRNP pathway), import of NL-15 RNA was strongly inhibited DNA templates and in vitro transcription DNA templates for in vitro transcription were generated by PCR by the lectin WGA (Figure 9), indicating that the RNA is amplification of RNA coding regions using appropriate primer pairs. brought into the nucleus complexed with an NLS-con- Templates used to transcribe U1, U2, U3 and U6 RNAs were described taining protein, as is U6 RNA (Fischer et al., 1991). previously (Terns et al., 1993, 1995). The template for U5 RNA was Furthermore, both the formation of complexes between generated by PCR amplification of the X.laevis X.l.U5 11H gene (Kazmaier et al., 1987) using a 5 primer containing the SP6 promoter NL-15 RNA and La in the cytoplasm, and the import of sequence (5-GGAATTCGATTTAGGTGACACTATAGAATACTCTG- NL-15 RNA into the nucleus were inhibited by cytoplasmic GTTTCT-3) and a 3 primer with a two nucleotide extension to make injection of antibodies that recognized La protein. These precursor length U5 RNA (5-AGTACCTGGTGTGAACCAGGC-3). results suggest that the complex responsible for import of The template for hY1 RNA was described previously (Simons et al., NL-15 RNA either contains La or requires La for its 1994). In vitro transcription of T7 or SP6 DNA templates was done in 20 μl reactions containing 40 mM Tris–HCl pH 7.9, 6 mM MgCl , formation. 2 2 mM spermidine, 10 mM NaCl, 0.1 mg/ml BSA, 10 mM DTT, 2–4 The failure of other La binding RNAs such as U6 units RNasin, 0.3 mM rATP, rCTP and rUTP, 0.1 mM rGTP plus 20 μCi (Figure 10C) and hY1 RNA (data not shown) to compete 32 7 [α- P]rGTP (25 pmol) and either 0.5 mM m GpppG or ApppG-cap for nuclear import of NL-15 RNA most likely is due to dinucleotide (NEB) or 2 mM γ-mpppG (kindly provided by Ram Reddy); incubation was for 1–2 h at 37°C with 20 units of either T7 or SP6 their inability to interact efficiently with La protein in the RNA polymerase. Unlabeled competitor RNAs were prepared in 100 μl cytoplasm. Both U6 and hY1 RNA normally bind La reactions containing 80 mM HEPES–KOH [N-(2-hydroxyethyl)piper- protein through their 3 uridylate stretch, but both RNAs azine-N-(2-ethanesulfonic acid)] pH 7.5, 16 mM MgCl , 2 mM spermi- are trimmed in the cytoplasm when injected at high levels. dine, 40 mM DTT, 2 mM rATP, rCTP, rUTP, 0.2 mM rGTP and 1 mM This results in the loss of their La binding site and (m GpppG) or 2 mM (γ-mpppG) cap analog. Incubation was for 2 h 803 C.Grimm, E.Lund and J.E.Dahlberg with 100 units of RNA polymerase, followed by a second addition of NL-15 RNA. Cleavage with RNase V1 (Pharmacia) was done with 0.7 100 units of RNA polymerase and further incubation for 2 h. All units of enzyme at 22°C in a 100 μl reaction containing 10 mM Tris RNA transcripts were purified by electrophoresis in a 8% denaturing (pH 7.5), 10 mM MgCl , 50 mM KCl, 10 μg tRNA and 50 fmol of NL- polyacrylamide gel and elution in 0.3 M NaCl, 10 mM Tris–HCl pH 7.6, 15 RNA. After 2, 6 and 18 min, 25 μl aliquots were removed and the 0.1 mM EDTA and 0.5% SDS. reactions were stopped by the addition of SDS (to a final concentration of 0.1%) and 10 μg yeast RNA. RNAs were prepared immediately by Oocyte injection and analysis of RNA transport phenol–chloroform (24:1) extraction and ethanol precipitation. Cleavage –4 Nuclei or cytoplasms of intact stage V and VI oocytes from X.laevis with RNase A (ICN) and RNase T1 (Calbiochem) was done with 10 were injected with 12 nl of H O containing 1–10 fmol of P-labeled units or 1 unit of enzyme, respectively for 18 min at 55°C in 7 M urea, RNAs and where indicated, different amounts of unlabeled competitor 1 mM EDTA, 25 mM Na-Acetate pH 7.0 and 10 μg tRNA. For controls, RNAs. The injection mixture also contained blue dextran to monitor the NL-15 RNA was incubated in buffer without enzyme under the respective accuracy of nuclear injection (Jarmolowski et al., 1994). After incubation conditions for 18 min. RNase cleavage products were separated on a at 18°C for different times (see figure legends), oocytes were manually 10% polyacrylamide gel containing 8.3 M urea. dissected under mineral oil (Lund and Paine, 1990) into nuclear and cytoplasmic fractions. After proteinase K digestion, total RNAs were Site directed mutagenesis of individual RNAs isolated from each fraction by two extractions with phenol–chloroform NL-25 and NL-15 RNAs were mutagenized by PCR amplification (24:1) and ethanol precipitations and purified RNAs were analyzed by using the following sets of primers. For NL-25/mut1: 5 primer SP6-U1 electrophoresis in 8% polyacrylamide gels containing 7 M urea. 5mut (5-GAATTCGATTTAGGTGACACTATAGAATACTATGGTG- GCAGGGG-3) and 3 primer CS536; for NL-25/mut2: 5 primer T7 In vivo selection SELEX and 3 primer NL-25/mut2 (5-ATCAGGGGAAAGCGCG- The DNA template used to transcribe the pool of RNAs for the first AACGCAGTCCACTACCAGAATACTATGGAAAGTCCTCAGGG- round of selection was prepared by annealing 50 pmol of an 87 nucleotide 3); for NL-25/mut12: 5 primer SP6-U1 5mut and 3 primer NL-25/ oligonucleotide (complementary to the RNA sequence shown in Figure mut2; for NL-15/5Ext: 5 primer SP6-NL-RS (5-GGAATTCGATTTA- 1A) to 250 pmol of a partially overlapping oligonucleotide containing GGTGACACTATAGAACTAGAGTACTGGGATACTTACCTGGCA- the T7 promoter sequence plus nucleotides 1–19 of the RNA shown in GGGG-3) and 3 primer CS536. PCR products were purified by Figure 1A (T7 SELEX: 5-AATGTCGACTAATACGACTCACTATA- electrophoresis in a 6% polyacrylamide gel and used for in vitro GGGATACTTACCTGGCAGG-3). After annealing at 60°C, the products transcription. were extended with Stoffel enzyme (Perkin Elmer) for1hat 60°C. Full-length double-stranded products were purified by electrophoresis in Antibodies, immunoprecipitations and immunodepletions 2,2,7 a 6% polyacrylamide gel. For generation of the starting pool of RNAs, Rabbit polyclonal antibodies against the m G- (Bringmann et al., 250 ng of the gel-purified template was transcribed with T7 RNA 1983; kindly provided by R.Lu¨hrmann) and the m G-cap (Munns et al., polymerase and RNAs were purified as described above. For the first 1982; kindly provided by T.Munns) were used to precipitate deproteinized round of selection, 50 fmol of the experimental RNAs were injected RNAs, mouse monoclonal antibodies against Sm proteins (mAb Y12, together with 1–2 fmol each of the control RNAs into nuclei or Lerner et al., 1981; kindly provided by J. Steitz) and anti-La antibodies cytoplasms of 50 oocytes. Theoretically, this corresponded to 2.510 from human patient sera (B-103, GO; kindly provided by D.Kenan and molecules and thus could contain all of the 1.110 different molecules J.Keene) were used to precipitate RNPs from nuclear and cytoplasmic that can be formed from a 20 nucleotide long randomized sequence. extracts and deplete nuclear extracts of La protein. Anti-CBP20 antibodies After 20–24 h of incubation at 18°C, oocytes were dissected into nuclei (rabbit; Izaurralde et al., 1995; kindly provided by E.Izaurralde and and cytoplasms and total RNA prepared from both compartments. I.Mattaj) were used to immunodeplete nuclear extract of CBC. Analytical polyacrylamide gels were used to determine the nucleo– Immunoprecipitations were done as described previously (Terns et al., cytoplasmic distribution of experimental and control RNAs at each round 1992). For the injection of anti-La antibodies, total IgGs were purified of selection. Prior to reverse transcription and PCR amplification (RT– from serum GO, essentially as described (Harlow and Lane, 1988). PCR), the experimental RNA in the nuclear fraction was size selected IgGs from 1.2 ml serum were bound to protein A–Sepharose beads in a and purified by electrophoresis in a 8% polyacrylamide gel containing 1.5 ml column in 100 mM Tris–HCl (pH 8). The column was washed 7 M urea. Reverse transcription was done in a 20 μl reaction containing with 10 column volumes of 100 mM Tris–HCl (pH 8) followed by 10 50 mM Tris–HCl pH 8.5, 75 mM KCl, 10 mM DTT, 3 mM MgCl , column volumes of 10 mM Tris (pH 8) and IgGs were eluted with 0.5 mM dNTPs, 2–4 units RNasin and 5 μM primer CS536 (5- 100 mM glycine (pH 3). After the addition of 0.1 volumes of1MTris– ATCAGGGGAAAGCGCGAACGCAGTCC-3). The mixture was HCl (pH 8), the neutralized IgGs were precipitated by the addition heated for 2 min at 95°C and cooled to 37°C prior to the addition of 1 of 1 volume of saturated (NH ) SO and collected by 30 min of 4 2 4 μl of M-MLV reverse transcriptase (200 units/μl, USB). After incubation centrifugation at 3000 g. The pellet was drained, resuspended in 0.1 ml for 15 min at 37°C, 80 μl of a mixture containing 1.8 mM MgCl , 50 of PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na HPO , 1.8 mM KH PO , 2 2 4 2 4 mM KCl, 10 mM Tris–HCl pH 9.0, 0.1% Triton X-100, 8 μM primer pH 7.4) and dialyzed against 3 2000 ml of PBS. IgGs were further T7 SELEX and 0.5 units of Taq DNA polymerase (Promega) were concentrated ~8-fold using a microconcentrator (microcon 100; Amicon). added and overlayed with mineral oil to prevent condensation. PCR Anti-La activity of the concentrated IgGs was tested in separate gel shift amplification was done using 35 cycles of denaturation (1 min at 95°C), experiments (data not shown) and 60 nl (per oocyte) of the solution annealing (45 s at 68°C) and extension (1 min at 72°C). RT–PCR containing 3 mM DTT and 4 units RNasin/μl were injected into the products were fractionated by electrophoresis in a 6% polyacrylamide cytoplasm of each oocyte. gel and the purified DNA templates were used to transcribe RNA for Anti-CBP20 or anti-La (GO) antibodies were coupled to protein A– the next round of selection. The total amounts of RNA used for injection Sepharose beads and used to immunodeplete nuclear extract from La were: 50 (rounds 1–4) or 2–10 (rounds 5–12) fmol per oocyte and the protein or CBC, respectively. Extract from 50 nuclei was incubated with number of oocytes injected were 50 (rounds 1–2), 30 (round 3) or 5–10 the respective antibodies for 1.5 h on ice with occasional stirring. The (rounds 4–12). The RT–PCR products after the 12th round of selection mixes were spun for 10 s and the supernatants used as immunode- were re-amplified using a 5 primer containing a HindIII restriction site pleted extracts. and a 3 primer containing a EcoRI restriction site. These two sites were used for cloning of the PCR products into pGEM-4Z vector (Promega). Complex formation and native gel electrophoresis Escherichia coli cells were transfected by electroporation and plasmids Nuclear extracts from oocytes were prepared as described (Terns et al., were isolated from individual colonies. Inserts were sequenced by the 1995). For complex formation, 10 fmol (1 μl) of m G-capped NL-15 or dideoxy termination method using Sequenase version 2.0 (USB). γ-mpppG-capped U6 RNA were mixed with 50 ng of 23S rRNA (1 μl) and 8 μl (0.5 oocyte equivalents) of nuclear extract in D buffer Structure probing (250 mM sucrose, 25 mM KCl, 5 mM MgCl ,3 mM DTT,50mM The substrate for structure probing in solution was in vitro prepared Tris–HCl, pH 7.6). After incubation for 20 min at 19°C, 2.5 μlof NL-15 RNA with a single label in the m G-cap structure. Unlabeled loading solution (50% glycerol, 2.5 mM EDTA, 0.01% bromphenol- 7 32 NL-15 RNA was m G-capped using guanylyltransferase plus [α- P] blue, 0.01% xylene cyanol) was added and the samples were fractionated GTP and S-adenosylmethionine (SAM) as described (Terns et al., 1995). immediately in native 6% polyacrylamide gels (30:0.8) in 0.5 TEB Cleavage with RNase One (Promega) was done with 0.03 units of (1 TEB: 90 mM Tris, 90 mM boric acid, 2.5 mM EDTA). For enzyme at 22°C in a 100 μl reaction containing 10 mM Tris–HCl pH supershifts, the samples with preformed complexes (see above) were 7.5, 5 mM EDTA, 200 mM Na-Acetate, 10 μg tRNA and 50 fmol of incubated for 20 min on ice with 1 μl anti-La antibodies B-103 (diluted 804 Nuclear localization of stable RNAs in D and mixed with RNasin) prior to addition of the loading solution. Davis,L.I. (1995) The nuclear pore complex. Annu. Rev. Biochem., 64, The gels were run at room temperature for ~2 h at 10 V/cm, fixed in a 865–896. solution containing 10% acetic acid and 20% methanol for 25 min and Eckner,R., Ellmeier,W. and Birnstiel,M.L. (1991) Mature mRNA 3 end dried prior to autoradiography. formation stimulates RNA export from the nucleus. EMBO J., 10, 3513–3522. Escherichia coli extracts containing human La protein Finlay,D.R., Newmeyer,D.D., Price,T.M. and Forbes,D.J. (1987) Escherichia coli cells [strain BL21(DE3) pLysS] expressing recombinant Inhibition of in vitro nuclear transport by a lectin that binds to nuclear human La protein were kindly provided by D.Kenan. Cells were grown pores. J. Cell Biol., 104, 189–200. in LB in a 50 ml culture; expression of La protein was induced by Fischer,U., Darzynkiewicz,E., Tahara,S.M., Dathan,N.A., Lu¨hrmann,R. the addition of IPTG (isopropyl β-D-thiogalactopyranoside) to a final and Mattaj,I.W. (1991) Diversity in the signals required for nuclear concentration of 0.4 mM. 3 h after induction, PMSF (phenylmethyl- accumulation of U snRNPs and variety in the pathways of nuclear sulfonyl fluoride) was added to a final concentration of 0.125 mg/ml transport. J. Cell Biol., 113, 705–714. and cells were harvested by centrifugation at 5000 g for 10 min at Fischer,U., Sumpter,V., Sekine,M., Satoh,T. and Lu¨hrmann,R. (1993) room temperature. Cells were resuspended in 2 ml of 25 mM Tris–HCl Nucleo-cytoplasmic transport of U snRNPs: definition of a nuclear pH 8.0, 3 mM MgCl , 0.1 mM EDTA, 0.5 mM DTT, 100 mM NaCl, location signal in the Sm core domain that binds a transport receptor 0.125 mg/ml PMSF and quickly frozen in a dry-ice ethanol bath. Cells independently of the m G-cap. EMBO J., 12, 573–583. were thawed in a 37°C water bath in the presence of freshly added Fischer,U., Meyer,S., Teufel,M., Heckel,C., Lu¨hrmann,R. and PMSF and sonicated for 2 30 s (Branson sonifier, setting 2). After Rautmann,G. (1994) Evidence that HIV-1 Rev directly promotes the centrifugation at 12 000 g for 30 min at 4°C, the cleared supernatant nuclear export of unspliced RNA. EMBO J., 13, 4105–4112. was collected and fresh PMSF added. The extract was stored at 4°C and Fischer,U., Huber,J., Boelens,W.C., Mattaj,I.W. and Lu¨hrmann,R. (1995) used as source for human La protein. Extracts from uninduced cells The HIV-1 Rev activation domain is a nuclear export signal that were prepared as above, except that no IPTG was added. 1 μl extract accesses an export pathway used by specific cellular RNAs. Cell, 82, (diluted 1:16 in D ) was used for complex formation as described 475–483. above. Forbes,D.J. (1992) Structure and function of the nuclear pore complex. Annu. Rev. Cell Biol., 8, 495–527. UV-crosslinking Go¨rlich,D. and Mattaj,I.W, (1996) Nucleocytoplasmic transport. Science, 10–30 fmol of RNA, labeled only in its 5 m G-cap (Terns et al., 1995), 271, 1513–1518. were incubated in nuclear extracts as for complex formation (see above) Grimm,C., Stefanovic,B. and Schu¨mperli,D. (1993) The low abundance in the absence or presence of unlabeled competitor RNAs as indicated of U7 snRNA is partly determined by its Sm binding site. EMBO J., in the figure legends. The extracts were either untreated or depleted 12, 1229–1238. from La protein or CBC. After 20 min of incubation, the samples were Grimm,C., Lund,E. and Dahlberg,J.E. (1995) In vivo selection of RNA irradiated with UV light (short wavelength) for 45 min on ice. The sequences involved in nucleocytoplasmic RNA trafficking. Nucleic samples were then treated with RNase T1 and RNase A for 1 h at 37°C. Acids Symp. Ser., 33, 34–36. Proteins were concentrated by the addition of 5 volumes of acetone and Hamm,J. and Mattaj,I.W. (1989) An abundant U6 snRNP found in germ centrifugation. 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Journal

The EMBO Journal – Springer Journals

Published: Feb 15, 1997

Keywords: La protein; nuclear localization elements (NLEs); RNA transport; selection of RNAs in vivo; Sm consensus site

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