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The Heterogeneous Nuclear Ribonucleoproteins I and K Interact with a Subset of the Ro Ribonucleoprotein-associated Y RNAs in Vitro and in Vivo

The Heterogeneous Nuclear Ribonucleoproteins I and K Interact with a Subset of the Ro... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 23, Issue of June 8, pp. 20711–20718, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Heterogeneous Nuclear Ribonucleoproteins I and K Interact with a Subset of the Ro Ribonucleoprotein-associated Y RNAs in Vitro and in Vivo* Received for publication, February 13, 2001 Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M101360200 Gusta ´ v Fabini‡§¶, Reinout Raijmakersi, Silvia Hayer‡§, Michael A. Fourauxi, Ger J. M. Pruijni, and Gu ¨ nter Steiner‡§** From the ‡Institute of Medical Biochemistry, University of Vienna, the Vienna Biocenter, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria, the §Division of Rheumatology, University Hospital of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria, and the iDepartment of Biochemistry, Nijmegen Center for Molecular Life Sciences, University of Nijmegen, NL-6500 HB Nijmegen, The Netherlands The hY RNAs are a group of four small cytoplasmic posed of one molecule of a small RNA and several proteins that RNAs of unknown function that are stably associated bind either directly to the RNA or indirectly via protein-protein with at least two proteins, Ro60 and La, to form Ro interactions (1, 2). Many of these complexes exert essential ribonucleoprotein complexes. Here we show that the functions that are often indispensable for survival, such as the heterogeneous nuclear ribonucleoproteins (hnRNP) I small nuclear RNPs, which are major components of the spli- and K are able to associate with a subset of hY RNAs in ceosomal machinery, or the signal recognition particle, which vitro and demonstrate these interactions to occur also in plays a key role in protein export. In contrast to these well vivo in a yeast three-hybrid system. Experiments defined complexes, the structure of the cytoplasmic Ro RNPs is performed in vitro and in vivo with deletion mutants of still not fully resolved, and their function has remained enig- hY1 RNA revealed its pyrimidine-rich central loop to be matic (3, 4). They are composed of one molecule of a small Y involved in interactions with both hnRNP I and K and RNA (transcribed by RNA polymerase III) and at least two clearly showed their binding sites to be different from proteins, the 60-kDa protein Ro60 and the 48-kDa phosphopro- the Ro60 binding site. Both hY1 and hY3 RNAs copre- tein La. However, although Ro60 and Y RNAs are present in cipitated with hnRNP I in immunoprecipitation comparable stochiometric amounts, La is ;50-fold more abun- experiments performed with HeLa S100 extracts and dant, and therefore the vast majority of La molecules is not cell extracts from COS-1 cells transiently transfected bound to Y RNAs, in contrast to Ro60 (5). with VSV-G-tagged hnRNP-I, respectively. Further- Y RNAs are highly conserved in evolution (6) and have been more, both anti-Ro60 and anti-La antibodies copre- found in all multicellular eukaryotic organisms and may also cipitated hnRNP I, whereas coprecipitation of hnRNP K be present in some bacteria (7) but, remarkably, have so far not was not observed. Taken together, these data strongly been detected in yeast. Interestingly, the genome of the nem- suggest that hnRNP I is a stable component of a atode Caenorhabditis elegans contains only one functional Y subpopulation of Ro RNPs, whereas hnRNP K may be RNA gene, whereas in humans and other vertebrates four transiently bound or interact only with (rare) Y RNAs that are devoid of Ro60 and La. Given that functions closely related Y RNA species exist. Unlike other RNA polym- related to translation regulation have been assigned to erase III transcripts, Y RNAs retain the oligo(U) stretch at both proteins and also to La, our findings may provide their 39 end that forms the binding site for La; therefore these novel clues toward understanding the role of Y RNAs RNAs remain permanently associated with La (5, 8). On the and their respective RNP complexes. other hand, nuclear Y RNAs do not seem to be associated with Ro60, and it was suggested that Ro RNPs assemble upon export to the cytoplasm (3, 9). Small ribonucleoprotein (RNP) complexes are usually com- Several functions have been proposed for La, including reg- ulation of RNA polymerase III transcription (10 –12), involve- ment in internal ribosome entry site-dependent viral and cel- * This work was supported by a grant from the Austrian Science lular translation (13, 14), and a role in the assembly of small Fund (project part no. 5 of the Special Research Project “Modulators of RNA Fate and Function”) and in part by the Netherlands Foundation nuclear RNPs (15), but it is still not entirely clear which role for Chemical Research with financial aid from the Netherlands Orga- this abundant nuclear and cytoplasmic protein plays in vivo. In nization for Scientific Research. The costs of publication of this article particular, it is completely unknown whether a unique function were defrayed in part by the payment of page charges. This article must can be attributed to Y RNA-associated La. Much less is known therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. about biological activities of Ro60 (4). In the cytoplasm it may Present address: Inst. of Chemistry, Div. of Glycobiology, Agricul- be involved in the regulation of translation as recently demon- tural University, Muthgasse 18, A-1190 Vienna, Austria. strated for ribosomal protein L4 in Xenopus laevis oocytes (16) ** To whom correspondence should be addressed: Div. of Rheumatol- and in the nucleus Ro60 may be implicated in a discard path- ogy, Dept. of Internal Medicine III, University Hospital of Vienna, way of 5 S rRNA by recognizing incorrectly processed and Waehringer Guertel 18-20, A-1090 Vienna, Austria. Tel.: 431-40400- 2121; Fax: 431-40400-4306; E-mail: [email protected]. misfolded molecules (17, 18). However, these (proposed) func- The abbreviations used are: RNP, ribonucleoprotein; hnRNP, het- tions do not seem to be dependent on the presence of Y RNAs in erogeneous nuclear ribonucleoprotein; IPP, immunoprecipitation buff- er; PTB, polypyrimidine tract-binding protein; SLE, systemic lupus erythematosus; VSV, vesicular stomatitis virus; b-gal, b-galactosidase; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; acid; PCR, polymerase chain reaction; X-gal, 5-bromo-4-chloro-3-indolyl CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic b-D-galactopyranoside. This paper is available on line at http://www.jbc.org 20711 This is an Open Access article under the CC BY license. 20712 Interaction of hnRNP I and K Proteins with hY RNAs 0.1% Nonidet P-40) for1hat room temperature, filters were incubated X. laevis nor in C. elegans. Interestingly, a prokaryotic homo- with monoclonal anti-VSV-G tag antibody (Roche Diagnostics, Almere, logue of Ro60 was recently discovered in the bacterium Deino- Netherlands) diluted 1:500 in washing buffer for1hat room tempera- coccus radiodurans that seemed to contribute to the remarka- ture. After washing, bound antibodies were detected by chemilumines- ble resistance of this organism to ultraviolet radiation (7). This cence using horseradish peroxidase-conjugated goat anti-mouse IgG as may lead to speculation about a RNA chaperoning function of secondary antibody (Dako, Glostrup, Denmark). Ro60 as has been recently proposed for La (19). Immunoprecipitation—Immunoprecipitations with monoclonal anti- bodies to Ro60, La, and hnRNP I and K were performed as described Although the stable association of Ro60 and La with Y RNAs with antibodies coupled to protein A-Sepharose beads (Amersham is beyond experimental doubt, the presence of other compo- Pharmacia Biotech) using dimethylpimelimidate (Sigma) as a cross- nents in addition to Ro60 and La is still a matter of debate. linking agent (28). Twenty ml of a HeLa cell extract was diluted in 0.5 Thus, the suggested associations of a 52-kDa protein (Ro52) ml of immunoprecipitation buffer (IPP)-150 (10 mM Tris-HCl, pH 7.5, and the Ca -binding protein calreticulin have remained con- 150 mM NaCl, 0.05% Nonidet P-40) and incubated for1hat4 °C with troversial (20 –25), and recently reported interactions of two immobilized antibodies (20 ml of packed bead volume). Subsequently, beads were washed three times with IPP-150 and resuspended in 400 ml novel proteins with Ro60 observed in yeast two- and three- of IPP-150 containing 0.5% SDS. RNA was extracted with phenol/ hybrid systems need to be confirmed (26, 27). In a previous chloroform, precipitated with ethanol using 10 mg of glycogen as carrier, study, we demonstrated in vitro binding of several proteins and analyzed by Northern blot hybridization. For isolation of proteins contained in a HeLa S100 extract to hY RNAs (28). These 200 ml of a HeLa cell extract was applied to 1 ml of anti-Ro or anti-La proteins (with molecular masses between 53 and 80 kDa) immunoaffinity columns using a peristaltic pump at a low flow rate. bound specifically to hY1 and hY3 RNA but only weakly or not After washing with at least 10 bed volumes of IPP-150, proteins were eluted with IPP-1000 (10 mM Tris-HCl, pH 7.5, 1000 mM NaCl, 0.05% at all to hY4 and hY5 RNA. Interestingly, autoantibodies to Nonidet P-40). these proteins were found in sera from patients with the rheu- For immunoprecipitations carried out with extracts from transfected matic autoimmune disease systemic lupus erythematosus cells, monoclonal antibodies (anti-VSV-G-tag, anti-Ro60, or anti-La) (SLE) who commonly develop autoantibodies to Ro and La (29). were coupled to protein A-agarose beads by incubating overnight at 4 °C Using deletion mutants of hY1 RNA we were able to show that in IPP-500 buffer (10 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.05% the binding sites for these proteins were distinct from the Ro60 Nonidet P-40). Subsequently, the antibody-coated beads were equili- brated with IPP-150 and incubated with cell extracts for2hat4 °C. binding site. In this report we demonstrate that two of these Then the beads were extensively washed with IPP-150, and coprecipi- proteins are identical to the heterogeneous nuclear RNP pro- tating RNAs were isolated by phenol/chloroform extraction and ethanol teins I (hnRNP I) and K (hnRNP K) and present evidence that precipitation. they are able to associate with a subset of hY RNAs in vivo. In Vitro Transcription of Y RNAs and Northern Blot Analyses—In vitro transcription of human sense and antisense hY RNAs and of hY1 EXPERIMENTAL PROCEDURES RNA deletion mutants was performed as described (28). To generate Sera and Antibodies—For immunodetection of proteins binding to biotinylated RNAs biotin 16-UTP (Roche Molecular Biochemicals) was hY1 and hY3 RNA in vitro, an anti-Ro positive serum from a patient used at 75 mM concentration, for preparation of radiolabeled RNAs 0.5 (BM) with SLE containing antibodies to these proteins was employed mM UTP and 40 mCi of [a- P]UTP (PerkinElmer Life Sciences) were (28). Sera from healthy persons and from patients with rheumatoid employed. The plasmid encoding 5 S rRNA was a kind gift of Dr. K. arthritis (which do not contain anti-Ro or anti-La autoantibodies) were Nierhaus (Max Planck Institute for Molecular Genetics, Berlin, used as negative controls. Monoclonal antibodies used were anti-La Germany). For detection of Y RNAs by Northern blot hybridization SW5 (30), anti-Ro60 2G10 (31), anti-hnRNP K 2G14 (Ref. 32; kind gift RNAs were separated on 10% polyacrylamide, 7 M urea gels, electro- of Gideon Dreyfuss, Howard Hughes Medical Institute, University of blotted onto nylon membranes (Zeta-probe; Bio-Rad), fixed by UV cross- Pennsylvania, Philadelphia, PA), anti-PTB 3 (Ref. 33; kind gift of D. M. linking, and hybridized with P-labeled antisense hY RNA transcripts Helfman, Cold Spring Harbor Laboratory), and an anti-human inter- by incubating overnight at 65 °C as described (28, 34). leukin-6 antibody (Janssen Biochimica, Denmark) as control. Reconstitution and Purification of hY RNPs—These procedures were Cellular Extracts—HeLa S100 extracts for use in reconstitution as- performed essentially as described (28). To dissociate existing com- says were prepared essentially as described (28). Briefly, 1 3 10 HeLa plexes, the salt concentration of the HeLa extract was first increased to cells (Computer Cell Culture Center, Mons, Belgium) were washed 1 M KCl followed by 30 min of incubation on ice. Then 10 mg of bioti- twice in isotonic buffer (10 mM Tris-HCl, pH 7.9, 140 mM KCl, 1.5 mM nylated hY RNA or biotinylated control RNA (5 S rRNA) and 200 mg MgCl ,1mM EDTA, 25% glycerol), resuspended in 2 pellet volumes of yeast tRNA (Roche) was added and the KCl concentration was read- buffer A (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1.5 mM MgCl ,1mM justed to 150 mM by diluting with reconstitution buffer (10 mM Tris- EDTA, 25% glycerol) and disrupted by Dounce homogenization. Nuclei HCl, pH 7.9, 2 mM MgCl ,1mM DTT, 5% glycerol). After incubating for were separated by brief centrifugation (3 min at 3,000 3 g), and the 20 min at 30 °C reconstituted hY RNP complexes were isolated by supernatant was first centrifuged for 20 min at 20,000 3 g and then for adding 20 ml of NeutrAvidin beads (Pierce) and rotating for1hat4°C. 1 h at 100,000 3 g. After measuring the protein concentration the S100 Beads were then washed five times with 1 ml of IPP-150, and bound extract was stored at 270 °C. proteins were isolated employing elution buffer (20 mM Tris-HCl, pH For immunoprecipitation experiments with transfected COS-1 cells, 7.9, 20 mM DTT, 2% SDS). Eluted proteins were heated for 5 min at total cell extracts were prepared after trypsinization, harvesting (800 65 °C and precipitated by adding 1 ml of glycogen (20 mg/ml) and 4 rpm, 5 min), and washing of the cells with ice-cold phosphate-buffered volumes of acetone. Samples were left at 270 °C for at least 30 min and saline (10 mM sodium phosphate, pH 7.4, 150 mM NaCl). Cells were subsequently centrifuged for 15 min at 15,000 3 g. Recovered proteins sonified with a Branson microtip (three times for 10 s at 4 °C) in lysis were dissolved either in SDS sample buffer for SDS-PAGE or in buffer (25 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM dithioerythritol, 2 two-dimensional lysis buffer for application in two-dimensional mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40). electrophoresis. After 15 min cell debris was removed by centrifugation (12,000 rpm, 15 Two-dimensional Gel Electrophoresis—Proteins binding to biotiny- min), and the extract was stored at 270 °C. lated hY RNAs in vitro were dissolved in 20 – 40 ml of lysis buffer Western Blot Analyses—Proteins were separated by SDS-PAGE and containing 9 M urea, 2% CHAPS (Sigma), 0.8% ampholyte pH 3–10 (40% subsequently blotted onto nitrocellulose as described (28, 34). The ni- solution, Fluka, Switzerland), 1% DTT. High resolution two-dimen- trocellulose sheets were blocked for 1 h with 3% nonfat dried milk in sional gel electrophoresis on immobilized pH gradients was carried out incubation buffer (10 mM Tris-HCl, pH 7.4, 140 mM NaCl, 0.1% Triton on ready-cut IPG Immobiline strips, pH 3–10 nonlinear, 18 cm long X-100) and subsequently incubated for 1 h either with human autoim- (Amersham Pharmacia Biotech). After overnight incubation in rehydra- mune serum diluted 1:50 or with monoclonal antibodies, respectively. tion buffer (8 M urea, 0.5% CHAPS, 15 mM DTT, 0.2% ampholyte pH After washing the nitrocellulose filters three times with incubation 3–10), the strips were focused on a horizontal electrophoresis apparatus buffer, bound antibodies were detected by alkaline phosphatase-conju- (Amersham Pharmacia Biotech) in a stepwise fashion: 0.5 h at 300 V, gated goat anti-human or anti-mouse IgG (Chemicon, Temecula, CA). 1hat500V,1hat 1500 V, and 15 h at 2500 V (total 40 kVh) at room Extracts from transfected cells were fractionated by SDS-PAGE and temperature. The IPG strips were subsequently incubated for 15 min in blotted onto a nitrocellulose filter. After blocking the filters in washing equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% buffer (phosphate-buffered saline containing 5% nonfat dried milk, SDS, traces of bromphenol blue) containing 10 mg/ml DTT and for 15 Interaction of hnRNP I and K Proteins with hY RNAs 20713 FIG.1. Characterization of hY1 RNA-binding proteins by two-dimensional gel electrophoresis and immunoblotting. Proteins from HeLa cell extracts binding to biotinylated hY1 RNA in reconstitution assays were separated by two-dimensional electrophoresis, transferred to a nitrocellulose membrane, and stained with serum BM from a patient with SLE recognizing several hY1 RNA-binding proteins in addition to Ro60 and La (marked by arrows). The two stained spots migrating below La presumably represent La degradation products. Positions of Ro60 and La were determined in a separate experiment using monoclonal antibodies to these proteins. The hnRNP K protein was identified by tandem mass spectrometry, the identity of hnRNP I (migrating as a doublet) was deduced from its characteristic position on the two-dimensional gel and immunologically confirmed using a monoclonal antibody against hnRNP I (see Fig. 2C). min in equilibration buffer containing 48 mg/ml iodoacetamide (Sigma). was verified by sequencing. The yeast strain L40uraMS2, which stably Equilibrated IPG strips were immersed in SDS running buffer for a few expresses the LexA DBD-MS2 coat protein was double transformed seconds and placed on top of a 1-mm vertical SDS 10% polyacrylamide with plasmids containing the hybrid RNAs and the hybrid proteins, gel. SDS-PAGE was run for 4 –5 h at 20 mA, and the gels were then respectively. Transformants were selected on synthetic medium plates stained with Coomassie Blue R-250 or electroblotted onto nitrocellulose lacking uracil and tryptophan. Expression of the bait proteins was membranes for immunodetection. Coomassie-stained spots correspond- checked by Western blotting using appropriate monoclonal antibodies. ing to proteins binding to hY1 RNA (and not to 5 S rRNA) were excised, Double transformants were then assayed for b-galactosidase (b-gal) completely destained, and subjected to sequence analysis by tandem expression on a filter using X-gal as a substrate and for growth on mass spectrometry performed after in gel trypsin digestion (Harvard selective medium without histidine. For quantitative determination of Microchemistry Facility, Cambridge, MA). b-gal activity cells were disrupted with glass beads, and, after deter- Transfection Experiments—VSV-G-tagged cDNAs were constructed mination of protein concentration, enzyme activity was measured pho- as follows. For the human La protein the N-terminal VSV-G tag tometrically at 420 nm using 2-nitrophenyl-b-D-galactopyranoside as a (MEIYTDIEMNRLGK) was introduced via PCR using the following substrate. As a positive control the established IRE-IRP interaction was primers: La-1 (59-GAATTCGCCACCATGGAGATTTATACAGACATA- used (36). GAGATGAACCGACTTGGAAAGCGCGGCCGCATGGCTGAAAATG- RESULTS GTGATAATG-39) and La-2 (59-CTCGAGCTACTGGTCTCCAGCACC- ATT-39) using a full-length La cDNA (35) as template. Indicated in bold Identification of Novel hY RNA-associated Proteins—Re- type are the introduced EcoRI, NotI, and XhoI sites. The VSV-G tag cently, we have described a set of five proteins with molecular encoding sequence is underlined. The PCR product was digested with masses between 53 and 80 kDa that in addition to Ro60 and La EcoRI/XhoI and cloned into the corresponding sites of the pcDNA3 bound to human hY RNAs in vitro, particularly to hY1 and hY3 vector (Invitrogen). The cDNAs encoding hnRNP K and hnRNP I were kindly provided by Gideon Dreyfuss (Howard Hughes Medical Institute, RNA (28). Interestingly, autoantibodies to these proteins were University of Pennsylvania, Philadelphia, PA). The human hnRNP K detected in several sera of patients with SLE who commonly cDNA was modified by PCR to introduce flanking NotI and XhoI sites, develop antibodies to Ro and La proteins. To further charac- allowing replacement of the La cDNA with the hnRNP K cDNA, which terize these hY RNA-binding proteins Ro RNP complexes were resulted in an N-terminally VSV-G-tagged hnRNP K construct in reconstituted in vitro by incubating HeLa S100 extracts with pcDNA3. An N-terminally Myc-tagged version of a human hnRNP I biotinylated hY1 RNA. Reconstitution reactions employing bi- cDNA in pcDNA3 was modified to replace the Myc tag with a VSV-G tag. The integrity of the constructs was confirmed by DNA sequencing. otinylated 5 S rRNA were performed in parallel to control for As a control, the empty pcDNA3 vector was used in transfection nonspecifically binding proteins. All reactions were supple- experiments. mented with a 20-fold excess of yeast tRNA as a nonspecific African green monkey cells (COS-1) were transiently transfected competitor. Reconstituted complexes were purified by NeutrA- with the expression constructs. Briefly, COS-1 cells were grown to 80% vidin affinity chromatography followed by two-dimensional gel confluency in Dulbecco’s modified Eagle’s medium supplemented with electrophoresis and subsequently analyzed by immunoblotting 10% heat-inactivated fetal calf serum and penicillin/streptomycin in 5% CO at 37 °C. Cells were trypsinized and resuspended in phosphate- using SLE serum BM known to recognize all five novel hY buffered saline. Approximately 5 3 10 cells were transfected with 20 RNA-binding proteins as well as Ro60 and La (Ref. 28; see also mg of plasmid DNA in a total volume of 400 ml of phosphate-buffered Fig. 2A). As can be seen in Fig. 1, several protein spots on the saline. Electroporation was performed at 300 V and a capacity of 125 membrane were stained by serum BM. The spots correspond- microfarads with a Gene Pulser II (Bio-Rad). Subsequently, the cells 2 ing to proteins binding specifically to hY1 RNA are indicated by were seeded in 75-cm culture flasks and cultured overnight. arrows. The positions of Ro60 and La with its typical isoelectric Yeast Three-hybrid System—To investigate the interaction of hnRNP I and hnRNP K with hY RNAs in vivo, a three-hybrid system (36) was isoforms (37–39) were confirmed in a separate experiment us- used (RNA-protein hybrid hunter kit; Invitrogen, Groningen, The Neth- ing monospecific sera and monoclonal antibodies to these pro- erlands). The DNAs encoding hY RNAs and hY1 RNA deletion mutants teins (not shown); the two spots visible below the La spots were introduced into the SmaI and AvrII sites of the pRH59 hybrid RNA presumably represent La degradation products. Apart from the vector downstream of two copies of the MS2 RNA sequence. hnRNP I spots corresponding to Ro60 and La proteins, four major pro- and hnRNP K hybrids were constructed by ligating the PCR amplified tein spots can be detected migrating above Ro60. The most fragments into the EcoRI and XhoI sites of the pYEASTrp2 hybrid protein vector. The sequence and orientation of all recombinant DNAs acidic of these proteins, which reproducibly migrated at ;68 20714 Interaction of hnRNP I and K Proteins with hY RNAs showed pronounced binding to hY1 RNA, but only hnRNP I efficiently bound to hY3 RNA, whereas the interaction of hnRNP K with hY3 RNA was relatively weak. In this assay, neither of the two proteins interacted detectably with hY4 or hY5 RNA, confirming previous data (28). To map the regions of hY1 RNA involved in binding of the two proteins, deletion mutants of (biotinylated) hY1 RNA were used. Truncation of the Ro60 binding site did not have any effect on binding of either hnRNP I or hnRNP K (Fig. 3B, lane 5), whereas mutation of the La binding site (39-terminal UU to AG) significantly decreased binding of both proteins (Fig. 3B, lane 6). The strongest effect was observed with a mutant lack- FIG.2. Detection of hnRNP K and hnRNP I in reconstituted hY1 RNP complexes. Proteins binding in reconstitution assays to ing the pyrimidine-rich central loop 2b, which bound hnRNP I biotinylated hY1 RNA (lanes 1) or 5 S rRNA (lanes 2) were isolated by very weakly and hnRNP K not at all (Fig. 3B, lane 7). Deletion streptavidine affinity chromatography, separated by SDS-PAGE, and of stem2-loop1 or of stem3-loop3 had no or only little effect on probed by Western blotting with patient serum BM (A), a monoclonal binding of hnRNP I, whereas binding of hnRNP K appeared to anti-hnRNP K antibody (B), and a monoclonal anti-hnRNP I antibody (C). be reduced by ;50 and 80%, respectively (Fig. 3B, lanes 8 and 9). Finally, the binding of both proteins appeared to be some- kDa, was excised from a Coomassie-stained two-dimensional what increased with a mutant lacking the stem4-loop4 region gel run in parallel, digested with trypsin, and microsequenced (Fig. 3B, lane 10). A comparable result was obtained with by tandem electrospray mass spectrometry. This analysis pro- 35 S-labeled hnRNP I and K proteins translated in vitro in a vided a 19-amino acid tryptic peptide sequence (GSYGDLGG- wheat germ system (data not shown). PIITTQVTIPK), which completely matched a sequence con- Taken together, these results (i) clearly confirmed the in tained in the human hnRNP K protein (residues 378 –396). vitro binding of hnRNP I and K to hY1 RNA; (ii) showed the This result was compatible with the migration of the proteins interaction of the two proteins with hY3 RNA to be slightly in our two-dimensional gels, which largely corresponded to the (hnRNP I) or considerably (hnRNP K) weaker than with hY1 molecular mass (66 kDa) and isoelectric point values (6.1– 6.4) RNA; (iii) demonstrated the central loop 2b to be indispensable reported for hnRNP K (40). for efficient binding of both proteins to hY1 RNA; (iv) suggested The second protein that we could identify was a basic protein that La but not Ro60 is required for efficient binding; and (v) visible as a 62/60-kDa doublet. Previous experiments had al- indicated that the binding sites for the two hnRNP proteins are ready suggested the presence of a 60-kDa protein in reconsti- closely spaced but not necessarily identical. tuted hY1 RNPs that comigrated with Ro60 in SDS-PAGE and Interaction of hnRNP I and K with Native hY RNAs—To appeared to have similar RNA binding properties as the 62- investigate whether and which hY RNAs are associated with kDa protein (28). Because of its migration behavior and char- the hnRNP I and K proteins in vivo, a HeLa cell extract was acteristic doublet appearance, we hypothesized that this pro- subjected to immunoprecipitation using specific monoclonal tein was hnRNP I, the PTB, which is known to migrate as a antibodies to hnRNP I, hnRNP K, Ro60, and La. RNAs were double band of ;60 kDa in SDS-PAGE and has a reported pI of isolated from the immunoprecipitates by phenol-chloroform ex- 8.5 (40, 41). traction and probed with radiolabeled antisense hY RNAs by To confirm the presence of hnRNP I and hnRNP K in recon- Northern blot hybridization. As shown in Fig. 4A, coprecipita- stituted complexes hY1 RNA-binding proteins were separated tion of hY1 and hY3 RNA by the anti-hnRNP I monoclonal by SDS-PAGE and stained with either serum BM or mono- antibody was clearly observed although at a lower level as clonal antibodies to hnRNP K or hnRNP I, respectively (Fig. 2, compared with the precipitates obtained with the anti-La and lanes 1). Compatible with our previously published data serum anti-Ro60 antibodies, which efficiently precipitated all four hY BM recognized proteins of 80, 68, 65, 62, and 53 kDa in addition RNAs. RNA bands visible just below hY1 RNA and hY3 RNA to Ro60 and La (28). In contrast to serum BM, the anti-hnRNP (lanes 1 and 3) presumably corresponded to previously reported K antibody stained solely the 68-kDa band, whereas the anti- degradation products of these two hY RNAs known as hY2 and hnRNP I antibody recognized two bands of 62 and 60 kDa. hY3* RNAs (22, 42), which was consistent with the lack of Neither the serum nor the monoclonal antibodies recognized a coprecipitation of these molecules by the anti-La antibodies. protein in the control lanes containing proteins binding to 5 S These results not only confirmed the specific binding of hnRNP rRNA (Fig. 2, lanes 2). I to hY1 and hY3 RNA observed in vitro but also strongly Of the three other hY1 RNA-binding proteins recognized by suggested that hnRNP I is associated with hY1 and hY3 RNA the serum, the 53-kDa protein was horizontally streaked and in vivo. In contrast, coprecipitation of hY RNAs by the anti- only weakly reactive with the serum on two-dimensional im- hnRNP K antibody was not detectable (lane 4). munoblots and could therefore not be sequenced. For the 65- Although these data suggested that the association of kDa protein migrating as a double spot at neutral pI and for the hnRNP I with hY1 and hY3 RNA occurs also in vivo, they did basic protein(s) migrating at ;70 –72 kDa (presumably comi- not allow us to conclude that this protein was present also in Ro grating with hnRNP K in SDS-PAGE) no unambiguous data RNPs (i.e. hY RNA complexes containing both Ro60 and La). To could be obtained, and the 80-kDa protein was not visible on address this question, Ro and La RNPs were isolated from a Coomassie-stained two-dimensional gels. Interaction of hnRNP I and hnRNP K with hY RNAs in HeLa cell extract using anti-Ro60 and anti-La microprepara- tive immunoaffinity columns. Proteins were eluted with 1 M Vitro—To study the interaction of hnRNP I and hnRNP K with hY RNAs in more detail, reconstitution reactions were per- NaCl, separated by SDS-PAGE, and identified by immunoblot- ting using monoclonal antibodies against hnRNP I and hnRNP formed with all four hY RNAs as well as with several deletion mutants of hY1 RNA. Proteins binding to hY RNAs were sep- K; proteins isolated from in vitro reconstituted hY1 RNPs arated by SDS-PAGE, blotted onto nitrocellulose membranes, served as controls (Fig. 4B, lane 1). In these experiments the and subsequently probed with monoclonal antibodies to presence of hnRNP I in both anti-La and anti-Ro60 eluates was hnRNP I and K as described above (Fig. 3); both proteins reproducibly observed (Fig. 4B, lanes 2 and 3), demonstrating Interaction of hnRNP I and K Proteins with hY RNAs 20715 FIG.3. Interaction of hnRNP K and hnRNP I with hY RNAs and hY1 RNA deletion mutants. Reconstitution reac- tions were performed with biotinylated hY RNAs and deletion mutants of hY1 RNA, and binding of hnRNP I and K was detected by immunoblotting employing monoclonal antibodies. Note that mutant DS1L1 lacks the Ro binding site and mu- tant DS1L1sty lacks the La binding site. A, proposed secondary structure of hu- man hY1 RNA and hY1 RNA deletion mu- tants. wt, wild type; S, stem; L, loop. B, Western blot analysis of hY RNA associ- ation with hnRNP K and hnRNP I. FIG.4. Association of hnRNP I with native hY RNAs and Ro RNPs in HeLa cell extracts. A, Northern blot analysis of hY RNAs immunoprecipitated from a HeLa cell extract by monoclonal antibodies to Ro60 (lane 1), La (lane 2), hnRNP I (lane 3), and hnRNP K (lane 4). A monoclonal antibody to human interleukin-6 was used as a negative control (lane 5), and 5% of the total input RNA served as a positive control (lane 6). B, Western blot analysis of hnRNP I immunoprecipitated by the anti-La (lane 2) and anti-Ro60 (lane 3) monoclonal antibodies; a monoclonal anti-interleukin-6 antibody served again as a negative control (lane 4). For comparison hY1 RNA-associated proteins isolated from in vitro reconstituted complexes are shown (lane 1). that this protein was contained in Ro RNP complexes. On the Yeast Three-hybrid System—To investigate the interactions of other hand, and in agreement with the RNA precipitation data, hnRNP I and hnRNP K with hY RNAs in a living cell, we made hnRNP K could not be detected in these eluates (not shown). use of the yeast three-hybrid system (36). In analogy to the To confirm these data and to account for the possibility that widely used two-hybrid system, this system is based on tran- the epitope recognized by the anti-hnRNP K monoclonal anti- scriptional activation of the reporter genes his3 and lacZ upon body might be inaccessible when the hnRNP K is complexed interaction of RNA with the protein of interest. In our case the with hY RNA, we expressed both hnRNP proteins and, as a first (protein) hybrid consisted of the DNA-binding domain of control, La as VSV-G-tagged fusion proteins in transiently the transcriptional activator LexA fused to the RNA-binding transfected COS-1 cells. Western blot analysis with a mono- viral MS2 coat protein (LexA DBD-MS2), the second (RNA) clonal antibody directed to the VSV-G tag demonstrated that hybrid consisted of hY RNA (or hY1 RNA deletion mutants) the three tagged proteins are expressed to similar amounts cloned downstream of the MS2 RNA sequence (MS2 RNA-hY (Fig. 5A). This antibody was then used to immunoprecipitate RNA), and the third (protein) hybrid was composed of the lysates of transfected cells that were subsequently analyzed for transcription activation domain of Gal4 fused to either hnRNP the presence of (coprecipitated) Y RNAs by Northern blot hy- I or hnRNP K (B42AD-hnRNP protein) (Fig. 6A). bridization. Also by this approach hnRNP I was found to asso- To examine the interaction of hY1 and hY3 RNA with ciate with Y1 and Y3 RNA (Fig. 5B, lane 5), and again no hnRNP I or hnRNP K, a yeast strain expressing the LexA association of hnRNP K with Y RNAs could be detected (Fig. DBD-MS2 coat protein hybrid was cotransformed with the 5B, lane 8). As expected, VSV-G-tagged La was associated with hybrid plasmids encoding these RNAs and proteins. Transfor- all four Y RNAs (lane 2), and no Y RNAs were detectable when mants lacking any of the (RNA or protein) hybrid components cells were transfected with an “empty vector” (lane 11). All four were not able to grow on a medium deficient of histidine (not Y RNAs were also coprecipitated by the anti-La (lane 3) and shown) and showed no or only little b-gal activity when grown anti-Ro60 (lanes 6, 9, and 12) antibodies. on a synthetic medium lacking uracil and tryptophan (Fig. 6B). Interaction of hnRNP I and K with hY1 and hY3 RNA in a In contrast, elevated b-gal activity was clearly seen in trans- 20716 Interaction of hnRNP I and K Proteins with hY RNAs this has not been presented yet (3, 4). Here we have identified two of the proteins previously described by us to associate with hY RNAs in vitro (28) as hnRNP I and hnRNP K. The two proteins interacted strongly with hY1 RNA and to a lesser extent with hY3 RNA but not with hY4 or hY5 RNA, which is in agreement with our previous findings. Immunoprecipitation experiments performed with extracts from HeLa cells and tran- siently transfected COS-1 cells provided substantial evidence for in vivo association of hnRNP I with hY1 and hY3 RNA and indicated the existence of a subpopulation of Ro RNPs contain- ing hnRNP I in addition to Ro60 and La. The observation that the anti-hnRNP I antibody precipitated significantly smaller amounts of hY RNAs than the anti-Ro60 antibody (although comparable amounts of Ro60 and hnRNP I appeared to be bound in the reconstitution assays) indicates that, in contrast to Ro60, hnRNP I is associated with a minor portion (10 –20%) of hY1 and hY3 RNAs. Although these assays did not provide any evidence for hY RNA-hnRNP K interactions in these cel- lular extracts, a clearly positive result was obtained in a yeast three-hybrid system that was comparable with that obtained with hnRNP I. Thus, hnRNP K may bind only to a rather minor subset of hY RNAs that are devoid of Ro60 (there is no Ro60 homologue in yeast) and therefore difficult to detect, or, alter- natively, the interaction with hY RNAs may have been dis- turbed during preparation of the cellular extracts or upon antibody binding in immunoprecipitation assays. The importance of an intact La binding site for efficient binding of hnRNP I and hnRNP K was remarkable and leads us FIG.5. Association of VSV-tagged hnRNP I with Y RNAs in to speculate that the function of La in hY RNP assembly may transiently transfected cells. COS-1 cells were transfected with constructs encoding VSV-G-tagged La, hnRNP I, and hnRNP K, and an be that of an RNA chaperone being required for correct folding empty vector (mock), and 24 h after transfection cell lysates were of hY RNAs, thus enabling binding of other proteins (with the prepared and analyzed by Western blotting (A) or Northern blotting (B). notable exception of Ro60). This would be consistent with re- A, expression of the VSV-G-tagged proteins La, hnRNP I, and hnRNP K analyzed by Western blotting using a monoclonal anti-VSV-G tag anti- cent reports on the chaperoning role of La in small nuclear body. The positions of the molecular mass markers are indicated on the RNP assembly and pre-tRNA processing (12, 15, 19). right. B, Northern blot analysis of Y RNAs coprecipitating with VSV- The results obtained with hY1 RNA deletion mutants G-tagged proteins. The lysates were subjected to immunoprecipitation showed that the internal pyrimidine loop (71– 86 nucleotides) with monoclonal anti-VSV-G tag, anti-La, or anti-Ro60 antibodies, re- was indispensable for efficient association of the two hnRNP spectively. RNA isolated from the total lysates (input) and from the immunoprecipitates was analyzed by Northern blot hybridization using proteins with hY1 RNA and demonstrated their binding sites to antisense hY RNAs as probes. The positions of Y1, Y3, Y4, and Y5 are be clearly different from the Ro60 binding site. Both hnRNP I indicated. The band indicated with ** probably represents a degrada- and K are known to bind to pyrimidine-rich sequences (43, 44). tion product of Y3, previously designated Y3** (42). Thus, hnRNP I, which is commonly known as PTB, binds to the polypyrimidine stretch present near the 39 splice site of many formants expressing hY1 RNA and hnRNP I or hnRNP K introns and also to pyrimidine-rich sequences of mature hybrids, respectively, whereas b-gal activity exceeded back- mRNAs (45). For hnRNP K, which shows increased affinity for ground levels only by approximately 2-fold in hY3 RNA trans- poly(rC) sequences but does not bind to the polypyrimidine formants (Fig. 6B). The observed interactions of hY1 RNA with tract, several cytosine-rich recognition motives have been de- the two hnRNP proteins in the yeast system were largely scribed, such as the CT element (CCCTCCCCA) of the c-myc consistent with the in vitro reconstitution data described gene (46) and CU repeats (CCCCACCCUCUUCCCC) present above. Interactions of hnRNP I with hY3 RNA, on the other in the 39-untranslated region of erythroid 15-lipoxygenase hand, were considerably weaker than in the in vitro binding mRNA (47). These sequences as well as those recognized by experiments. hnRNP I (PTB) show significant similarities with the central When we examined binding of hnRNP I and K to hY1 RNA loop 2b region of hY1 RNA (UACUCUUUCCCCCCUU), sup- deletion mutants (same as shown in Fig. 3), mutant Y1DL2b porting the assumption that this region may directly interact lacking the pyrimidine-rich domain showed only low levels of with both proteins as suggested by the results obtained with b-gal activity when cotransformed with either hnRNP K or the deletion mutants. Remarkably, an internal pyrimidine-rich hnRNP I, again compatible with the in vitro results. In con- loop is present also in hY3 RNA, whereas it is lacking in hY4 trast, b-gal activity of the Y1DS3L3 mutant strain was even and hY5 RNA, which would be consistent with their failure to somewhat greater than that of wild-type Y1 RNA for both bind the two hnRNP proteins. hnRNP I and K, whereas deletion of stem4-loop4 (Y1DS4L4) The heterogeneous nature of Ro RNPs has been postulated led to a slightly decreased activation of the lacZ gene with both by several researchers based on biochemical fractionation and hnRNP proteins. The other two hY1 RNA mutants showed in vitro reconstitution data (22, 23, 28). The number of Y RNAs similar b-gal activities as wild-type hY1 RNA. has increased throughout the evolution from C. elegans, which DISCUSSION expresses only one Y RNA, to vertebrates with four Y RNAs (6, Although data from several laboratories suggest that Ro 48). Interestingly, the Ro60 binding site and the pyrimidine- RNP complexes may contain a number of other proteins in rich loop sequence represent the most conserved regions, being addition to Ro60 and La (20 –28), unambiguous evidence for already present in C. elegans RNA, which shows the greatest Interaction of hnRNP I and K Proteins with hY RNAs 20717 FIG.6. Interaction of hnRNP I and hnRNP K with hY1 and hY3 RNA in a yeast three-hybrid system. The yeast three-hybrid system was used to study interaction of the two hnRNP proteins with hY RNAs in vivo by measuring b-gal activity of transformed cells. A, schematic diagram showing gene activation by spe- cific interaction between hY-RNA and hnRNP I or K proteins in the yeast three- hybrid system. Expression of reporter genes lacZ and his3 is activated upon pos- itive interaction between hnRNP I or K and hY1 RNA dragging the following two domains of the transcriptional activator to a close vicinity: the DNA-binding do- main of LexA (DBD) and the activation domain of Gal 4 (AD). B, analysis of b-gal activity. Transformants were grown on synthetic medium lacking uracil and tryptophan, and b-gal activity was tested in the blue color X-gal filter assay (right) and quantitatively determined by direct measurement of the enzymatic activity in units/mg yeast mass (left). homology to hY3 RNA (49). This strongly suggests that the shown to bind specifically to several viral internal ribosome pyrimidine-rich element is important for Y RNA functioning, entry sites, thereby promoting ribosome binding in a cap-inde- including interactions with other proteins and/or RNAs. Fur- pendent manner (62– 64). Remarkably, a similar activity was thermore, the internal loop of hY1 RNA was shown to be also found for La (13), and cooperation of these two proteins in resistant to enzymatic and chemical cleavage (50), an observa- the regulation of internal ribosome entry site-dependent trans- tion also made with pyrimidine-rich loops of other small RNAs lation has been recently suggested (65). Furthermore, these transcribed by RNA polymerase III (51–53). Because the four proteins may be also required for efficient and correct initiation vertebrate Y RNAs show a high diversity in the central region, of cap-dependent translation by inhibiting translation of un- this part of the molecule might determine the fate (i.e. local- capped mRNAs (66). Apart from its role as transcription factor ization and function) of Y RNPs or Ro RNPs, respectively, (44, 67, 68), hnRNP K protein has been demonstrated to act as because of differential association with proteins of (more or a differentiation regulator by virtue of its binding to the control less) diverse function (such as hnRNP I and hnRNP K). elements at the 39 end of the erythroid 15-lipoxygenase mRNA, Several RNA polymerase III transcripts including RNase P thereby inhibiting translation (47). In a similar manner trans- RNA, RNase MRP RNA, and hY RNAs (except hY4 RNA) have lation of papilloma virus late mRNAs is inhibited by hnRNP K been localized to the perinucleolar compartments together with and the poly(rC)-binding protein (69). Furthermore, two trans- two hnRNP proteins, hnRNP I (41, 54), and CUG-binding pro- lation-related functions have been assigned to the Ro60 pro- tein/hNab50 (55, 56). Importantly, the presence of hnRNP I in tein, namely participation in a degradation pathway for mis- the perinucleolar compartments was found to be sensitive to folded 5 S rRNA (17) and a role in the regulation of translation RNase A treatment (57), which may be considered a further of ribosomal protein L4 in concert with La and cellular nucleic (though rather indirect) indication for in vivo interactions of acid-binding protein by binding to a polypyrimidine stretch in this protein with hY1 and hY3 RNAs. Remarkably, the pres- the 59-untranslated region of L4 mRNA (16, 70). ence of Ro60 and La could not be detected within the pe- Combining our experimental data and those of others one rinucleolar compartments (54), which may suggest the existence may speculate about the cellular role of Ro RNPs or hY RNPs, of hY RNA complexes containing hnRNP I (as well as other respectively: newly transcribed hY RNAs associate with La and proteins yet to be identified) but devoid of both Ro60 and La. move through the nucleoplasm toward the nuclear membrane hnRNP I and K are both shuttling proteins that belong to a where Ro60 associates just prior to export into the cytoplasm, group of multifunctional RNA-binding proteins exerting regu- being indispensable for nuclear export of hY RNAs (3, 9). Dur- latory roles at the post-transcriptional level, including RNA ing their intranuclear migration subsets of hY RNPs are processing and export as well as regulation of mRNA stability formed by virtue of association with various proteins including and translation (44, 58 – 61). Given that Ro RNPs are predom- hnRNP I and K, Ro52, and presumably other proteins includ- inantly, if not exclusively, localized in the cytoplasm, it is ing those reported to interact with hY RNA in vitro or with important to mention reported functions of hnRNP I and K that Ro60 in yeast two- and three-hybrid systems (26 –28). All these are related to translational events. Thus, hnRNP I has been proteins may be multifunctional and upon binding to hY RNAs 20718 Interaction of hnRNP I and K Proteins with hY RNAs 28. Fabini, G., Rutjes, S. A., Zimmermann, C., Pruijn, G. J. M., and Steiner, G. become destined to exert specific functions either in the nu- (2000) Eur. J. 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The Heterogeneous Nuclear Ribonucleoproteins I and K Interact with a Subset of the Ro Ribonucleoprotein-associated Y RNAs in Vitro and in Vivo

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 23, Issue of June 8, pp. 20711–20718, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Heterogeneous Nuclear Ribonucleoproteins I and K Interact with a Subset of the Ro Ribonucleoprotein-associated Y RNAs in Vitro and in Vivo* Received for publication, February 13, 2001 Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M101360200 Gusta ´ v Fabini‡§¶, Reinout Raijmakersi, Silvia Hayer‡§, Michael A. Fourauxi, Ger J. M. Pruijni, and Gu ¨ nter Steiner‡§** From the ‡Institute of Medical Biochemistry, University of Vienna, the Vienna Biocenter, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria, the §Division of Rheumatology, University Hospital of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria, and the iDepartment of Biochemistry, Nijmegen Center for Molecular Life Sciences, University of Nijmegen, NL-6500 HB Nijmegen, The Netherlands The hY RNAs are a group of four small cytoplasmic posed of one molecule of a small RNA and several proteins that RNAs of unknown function that are stably associated bind either directly to the RNA or indirectly via protein-protein with at least two proteins, Ro60 and La, to form Ro interactions (1, 2). Many of these complexes exert essential ribonucleoprotein complexes. Here we show that the functions that are often indispensable for survival, such as the heterogeneous nuclear ribonucleoproteins (hnRNP) I small nuclear RNPs, which are major components of the spli- and K are able to associate with a subset of hY RNAs in ceosomal machinery, or the signal recognition particle, which vitro and demonstrate these interactions to occur also in plays a key role in protein export. In contrast to these well vivo in a yeast three-hybrid system. Experiments defined complexes, the structure of the cytoplasmic Ro RNPs is performed in vitro and in vivo with deletion mutants of still not fully resolved, and their function has remained enig- hY1 RNA revealed its pyrimidine-rich central loop to be matic (3, 4). They are composed of one molecule of a small Y involved in interactions with both hnRNP I and K and RNA (transcribed by RNA polymerase III) and at least two clearly showed their binding sites to be different from proteins, the 60-kDa protein Ro60 and the 48-kDa phosphopro- the Ro60 binding site. Both hY1 and hY3 RNAs copre- tein La. However, although Ro60 and Y RNAs are present in cipitated with hnRNP I in immunoprecipitation comparable stochiometric amounts, La is ;50-fold more abun- experiments performed with HeLa S100 extracts and dant, and therefore the vast majority of La molecules is not cell extracts from COS-1 cells transiently transfected bound to Y RNAs, in contrast to Ro60 (5). with VSV-G-tagged hnRNP-I, respectively. Further- Y RNAs are highly conserved in evolution (6) and have been more, both anti-Ro60 and anti-La antibodies copre- found in all multicellular eukaryotic organisms and may also cipitated hnRNP I, whereas coprecipitation of hnRNP K be present in some bacteria (7) but, remarkably, have so far not was not observed. Taken together, these data strongly been detected in yeast. Interestingly, the genome of the nem- suggest that hnRNP I is a stable component of a atode Caenorhabditis elegans contains only one functional Y subpopulation of Ro RNPs, whereas hnRNP K may be RNA gene, whereas in humans and other vertebrates four transiently bound or interact only with (rare) Y RNAs that are devoid of Ro60 and La. Given that functions closely related Y RNA species exist. Unlike other RNA polym- related to translation regulation have been assigned to erase III transcripts, Y RNAs retain the oligo(U) stretch at both proteins and also to La, our findings may provide their 39 end that forms the binding site for La; therefore these novel clues toward understanding the role of Y RNAs RNAs remain permanently associated with La (5, 8). On the and their respective RNP complexes. other hand, nuclear Y RNAs do not seem to be associated with Ro60, and it was suggested that Ro RNPs assemble upon export to the cytoplasm (3, 9). Small ribonucleoprotein (RNP) complexes are usually com- Several functions have been proposed for La, including reg- ulation of RNA polymerase III transcription (10 –12), involve- ment in internal ribosome entry site-dependent viral and cel- * This work was supported by a grant from the Austrian Science lular translation (13, 14), and a role in the assembly of small Fund (project part no. 5 of the Special Research Project “Modulators of RNA Fate and Function”) and in part by the Netherlands Foundation nuclear RNPs (15), but it is still not entirely clear which role for Chemical Research with financial aid from the Netherlands Orga- this abundant nuclear and cytoplasmic protein plays in vivo. In nization for Scientific Research. The costs of publication of this article particular, it is completely unknown whether a unique function were defrayed in part by the payment of page charges. This article must can be attributed to Y RNA-associated La. Much less is known therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. about biological activities of Ro60 (4). In the cytoplasm it may Present address: Inst. of Chemistry, Div. of Glycobiology, Agricul- be involved in the regulation of translation as recently demon- tural University, Muthgasse 18, A-1190 Vienna, Austria. strated for ribosomal protein L4 in Xenopus laevis oocytes (16) ** To whom correspondence should be addressed: Div. of Rheumatol- and in the nucleus Ro60 may be implicated in a discard path- ogy, Dept. of Internal Medicine III, University Hospital of Vienna, way of 5 S rRNA by recognizing incorrectly processed and Waehringer Guertel 18-20, A-1090 Vienna, Austria. Tel.: 431-40400- 2121; Fax: 431-40400-4306; E-mail: [email protected]. misfolded molecules (17, 18). However, these (proposed) func- The abbreviations used are: RNP, ribonucleoprotein; hnRNP, het- tions do not seem to be dependent on the presence of Y RNAs in erogeneous nuclear ribonucleoprotein; IPP, immunoprecipitation buff- er; PTB, polypyrimidine tract-binding protein; SLE, systemic lupus erythematosus; VSV, vesicular stomatitis virus; b-gal, b-galactosidase; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; acid; PCR, polymerase chain reaction; X-gal, 5-bromo-4-chloro-3-indolyl CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic b-D-galactopyranoside. This paper is available on line at http://www.jbc.org 20711 This is an Open Access article under the CC BY license. 20712 Interaction of hnRNP I and K Proteins with hY RNAs 0.1% Nonidet P-40) for1hat room temperature, filters were incubated X. laevis nor in C. elegans. Interestingly, a prokaryotic homo- with monoclonal anti-VSV-G tag antibody (Roche Diagnostics, Almere, logue of Ro60 was recently discovered in the bacterium Deino- Netherlands) diluted 1:500 in washing buffer for1hat room tempera- coccus radiodurans that seemed to contribute to the remarka- ture. After washing, bound antibodies were detected by chemilumines- ble resistance of this organism to ultraviolet radiation (7). This cence using horseradish peroxidase-conjugated goat anti-mouse IgG as may lead to speculation about a RNA chaperoning function of secondary antibody (Dako, Glostrup, Denmark). Ro60 as has been recently proposed for La (19). Immunoprecipitation—Immunoprecipitations with monoclonal anti- bodies to Ro60, La, and hnRNP I and K were performed as described Although the stable association of Ro60 and La with Y RNAs with antibodies coupled to protein A-Sepharose beads (Amersham is beyond experimental doubt, the presence of other compo- Pharmacia Biotech) using dimethylpimelimidate (Sigma) as a cross- nents in addition to Ro60 and La is still a matter of debate. linking agent (28). Twenty ml of a HeLa cell extract was diluted in 0.5 Thus, the suggested associations of a 52-kDa protein (Ro52) ml of immunoprecipitation buffer (IPP)-150 (10 mM Tris-HCl, pH 7.5, and the Ca -binding protein calreticulin have remained con- 150 mM NaCl, 0.05% Nonidet P-40) and incubated for1hat4 °C with troversial (20 –25), and recently reported interactions of two immobilized antibodies (20 ml of packed bead volume). Subsequently, beads were washed three times with IPP-150 and resuspended in 400 ml novel proteins with Ro60 observed in yeast two- and three- of IPP-150 containing 0.5% SDS. RNA was extracted with phenol/ hybrid systems need to be confirmed (26, 27). In a previous chloroform, precipitated with ethanol using 10 mg of glycogen as carrier, study, we demonstrated in vitro binding of several proteins and analyzed by Northern blot hybridization. For isolation of proteins contained in a HeLa S100 extract to hY RNAs (28). These 200 ml of a HeLa cell extract was applied to 1 ml of anti-Ro or anti-La proteins (with molecular masses between 53 and 80 kDa) immunoaffinity columns using a peristaltic pump at a low flow rate. bound specifically to hY1 and hY3 RNA but only weakly or not After washing with at least 10 bed volumes of IPP-150, proteins were eluted with IPP-1000 (10 mM Tris-HCl, pH 7.5, 1000 mM NaCl, 0.05% at all to hY4 and hY5 RNA. Interestingly, autoantibodies to Nonidet P-40). these proteins were found in sera from patients with the rheu- For immunoprecipitations carried out with extracts from transfected matic autoimmune disease systemic lupus erythematosus cells, monoclonal antibodies (anti-VSV-G-tag, anti-Ro60, or anti-La) (SLE) who commonly develop autoantibodies to Ro and La (29). were coupled to protein A-agarose beads by incubating overnight at 4 °C Using deletion mutants of hY1 RNA we were able to show that in IPP-500 buffer (10 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.05% the binding sites for these proteins were distinct from the Ro60 Nonidet P-40). Subsequently, the antibody-coated beads were equili- brated with IPP-150 and incubated with cell extracts for2hat4 °C. binding site. In this report we demonstrate that two of these Then the beads were extensively washed with IPP-150, and coprecipi- proteins are identical to the heterogeneous nuclear RNP pro- tating RNAs were isolated by phenol/chloroform extraction and ethanol teins I (hnRNP I) and K (hnRNP K) and present evidence that precipitation. they are able to associate with a subset of hY RNAs in vivo. In Vitro Transcription of Y RNAs and Northern Blot Analyses—In vitro transcription of human sense and antisense hY RNAs and of hY1 EXPERIMENTAL PROCEDURES RNA deletion mutants was performed as described (28). To generate Sera and Antibodies—For immunodetection of proteins binding to biotinylated RNAs biotin 16-UTP (Roche Molecular Biochemicals) was hY1 and hY3 RNA in vitro, an anti-Ro positive serum from a patient used at 75 mM concentration, for preparation of radiolabeled RNAs 0.5 (BM) with SLE containing antibodies to these proteins was employed mM UTP and 40 mCi of [a- P]UTP (PerkinElmer Life Sciences) were (28). Sera from healthy persons and from patients with rheumatoid employed. The plasmid encoding 5 S rRNA was a kind gift of Dr. K. arthritis (which do not contain anti-Ro or anti-La autoantibodies) were Nierhaus (Max Planck Institute for Molecular Genetics, Berlin, used as negative controls. Monoclonal antibodies used were anti-La Germany). For detection of Y RNAs by Northern blot hybridization SW5 (30), anti-Ro60 2G10 (31), anti-hnRNP K 2G14 (Ref. 32; kind gift RNAs were separated on 10% polyacrylamide, 7 M urea gels, electro- of Gideon Dreyfuss, Howard Hughes Medical Institute, University of blotted onto nylon membranes (Zeta-probe; Bio-Rad), fixed by UV cross- Pennsylvania, Philadelphia, PA), anti-PTB 3 (Ref. 33; kind gift of D. M. linking, and hybridized with P-labeled antisense hY RNA transcripts Helfman, Cold Spring Harbor Laboratory), and an anti-human inter- by incubating overnight at 65 °C as described (28, 34). leukin-6 antibody (Janssen Biochimica, Denmark) as control. Reconstitution and Purification of hY RNPs—These procedures were Cellular Extracts—HeLa S100 extracts for use in reconstitution as- performed essentially as described (28). To dissociate existing com- says were prepared essentially as described (28). Briefly, 1 3 10 HeLa plexes, the salt concentration of the HeLa extract was first increased to cells (Computer Cell Culture Center, Mons, Belgium) were washed 1 M KCl followed by 30 min of incubation on ice. Then 10 mg of bioti- twice in isotonic buffer (10 mM Tris-HCl, pH 7.9, 140 mM KCl, 1.5 mM nylated hY RNA or biotinylated control RNA (5 S rRNA) and 200 mg MgCl ,1mM EDTA, 25% glycerol), resuspended in 2 pellet volumes of yeast tRNA (Roche) was added and the KCl concentration was read- buffer A (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1.5 mM MgCl ,1mM justed to 150 mM by diluting with reconstitution buffer (10 mM Tris- EDTA, 25% glycerol) and disrupted by Dounce homogenization. Nuclei HCl, pH 7.9, 2 mM MgCl ,1mM DTT, 5% glycerol). After incubating for were separated by brief centrifugation (3 min at 3,000 3 g), and the 20 min at 30 °C reconstituted hY RNP complexes were isolated by supernatant was first centrifuged for 20 min at 20,000 3 g and then for adding 20 ml of NeutrAvidin beads (Pierce) and rotating for1hat4°C. 1 h at 100,000 3 g. After measuring the protein concentration the S100 Beads were then washed five times with 1 ml of IPP-150, and bound extract was stored at 270 °C. proteins were isolated employing elution buffer (20 mM Tris-HCl, pH For immunoprecipitation experiments with transfected COS-1 cells, 7.9, 20 mM DTT, 2% SDS). Eluted proteins were heated for 5 min at total cell extracts were prepared after trypsinization, harvesting (800 65 °C and precipitated by adding 1 ml of glycogen (20 mg/ml) and 4 rpm, 5 min), and washing of the cells with ice-cold phosphate-buffered volumes of acetone. Samples were left at 270 °C for at least 30 min and saline (10 mM sodium phosphate, pH 7.4, 150 mM NaCl). Cells were subsequently centrifuged for 15 min at 15,000 3 g. Recovered proteins sonified with a Branson microtip (three times for 10 s at 4 °C) in lysis were dissolved either in SDS sample buffer for SDS-PAGE or in buffer (25 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM dithioerythritol, 2 two-dimensional lysis buffer for application in two-dimensional mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40). electrophoresis. After 15 min cell debris was removed by centrifugation (12,000 rpm, 15 Two-dimensional Gel Electrophoresis—Proteins binding to biotiny- min), and the extract was stored at 270 °C. lated hY RNAs in vitro were dissolved in 20 – 40 ml of lysis buffer Western Blot Analyses—Proteins were separated by SDS-PAGE and containing 9 M urea, 2% CHAPS (Sigma), 0.8% ampholyte pH 3–10 (40% subsequently blotted onto nitrocellulose as described (28, 34). The ni- solution, Fluka, Switzerland), 1% DTT. High resolution two-dimen- trocellulose sheets were blocked for 1 h with 3% nonfat dried milk in sional gel electrophoresis on immobilized pH gradients was carried out incubation buffer (10 mM Tris-HCl, pH 7.4, 140 mM NaCl, 0.1% Triton on ready-cut IPG Immobiline strips, pH 3–10 nonlinear, 18 cm long X-100) and subsequently incubated for 1 h either with human autoim- (Amersham Pharmacia Biotech). After overnight incubation in rehydra- mune serum diluted 1:50 or with monoclonal antibodies, respectively. tion buffer (8 M urea, 0.5% CHAPS, 15 mM DTT, 0.2% ampholyte pH After washing the nitrocellulose filters three times with incubation 3–10), the strips were focused on a horizontal electrophoresis apparatus buffer, bound antibodies were detected by alkaline phosphatase-conju- (Amersham Pharmacia Biotech) in a stepwise fashion: 0.5 h at 300 V, gated goat anti-human or anti-mouse IgG (Chemicon, Temecula, CA). 1hat500V,1hat 1500 V, and 15 h at 2500 V (total 40 kVh) at room Extracts from transfected cells were fractionated by SDS-PAGE and temperature. The IPG strips were subsequently incubated for 15 min in blotted onto a nitrocellulose filter. After blocking the filters in washing equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% buffer (phosphate-buffered saline containing 5% nonfat dried milk, SDS, traces of bromphenol blue) containing 10 mg/ml DTT and for 15 Interaction of hnRNP I and K Proteins with hY RNAs 20713 FIG.1. Characterization of hY1 RNA-binding proteins by two-dimensional gel electrophoresis and immunoblotting. Proteins from HeLa cell extracts binding to biotinylated hY1 RNA in reconstitution assays were separated by two-dimensional electrophoresis, transferred to a nitrocellulose membrane, and stained with serum BM from a patient with SLE recognizing several hY1 RNA-binding proteins in addition to Ro60 and La (marked by arrows). The two stained spots migrating below La presumably represent La degradation products. Positions of Ro60 and La were determined in a separate experiment using monoclonal antibodies to these proteins. The hnRNP K protein was identified by tandem mass spectrometry, the identity of hnRNP I (migrating as a doublet) was deduced from its characteristic position on the two-dimensional gel and immunologically confirmed using a monoclonal antibody against hnRNP I (see Fig. 2C). min in equilibration buffer containing 48 mg/ml iodoacetamide (Sigma). was verified by sequencing. The yeast strain L40uraMS2, which stably Equilibrated IPG strips were immersed in SDS running buffer for a few expresses the LexA DBD-MS2 coat protein was double transformed seconds and placed on top of a 1-mm vertical SDS 10% polyacrylamide with plasmids containing the hybrid RNAs and the hybrid proteins, gel. SDS-PAGE was run for 4 –5 h at 20 mA, and the gels were then respectively. Transformants were selected on synthetic medium plates stained with Coomassie Blue R-250 or electroblotted onto nitrocellulose lacking uracil and tryptophan. Expression of the bait proteins was membranes for immunodetection. Coomassie-stained spots correspond- checked by Western blotting using appropriate monoclonal antibodies. ing to proteins binding to hY1 RNA (and not to 5 S rRNA) were excised, Double transformants were then assayed for b-galactosidase (b-gal) completely destained, and subjected to sequence analysis by tandem expression on a filter using X-gal as a substrate and for growth on mass spectrometry performed after in gel trypsin digestion (Harvard selective medium without histidine. For quantitative determination of Microchemistry Facility, Cambridge, MA). b-gal activity cells were disrupted with glass beads, and, after deter- Transfection Experiments—VSV-G-tagged cDNAs were constructed mination of protein concentration, enzyme activity was measured pho- as follows. For the human La protein the N-terminal VSV-G tag tometrically at 420 nm using 2-nitrophenyl-b-D-galactopyranoside as a (MEIYTDIEMNRLGK) was introduced via PCR using the following substrate. As a positive control the established IRE-IRP interaction was primers: La-1 (59-GAATTCGCCACCATGGAGATTTATACAGACATA- used (36). GAGATGAACCGACTTGGAAAGCGCGGCCGCATGGCTGAAAATG- RESULTS GTGATAATG-39) and La-2 (59-CTCGAGCTACTGGTCTCCAGCACC- ATT-39) using a full-length La cDNA (35) as template. Indicated in bold Identification of Novel hY RNA-associated Proteins—Re- type are the introduced EcoRI, NotI, and XhoI sites. The VSV-G tag cently, we have described a set of five proteins with molecular encoding sequence is underlined. The PCR product was digested with masses between 53 and 80 kDa that in addition to Ro60 and La EcoRI/XhoI and cloned into the corresponding sites of the pcDNA3 bound to human hY RNAs in vitro, particularly to hY1 and hY3 vector (Invitrogen). The cDNAs encoding hnRNP K and hnRNP I were kindly provided by Gideon Dreyfuss (Howard Hughes Medical Institute, RNA (28). Interestingly, autoantibodies to these proteins were University of Pennsylvania, Philadelphia, PA). The human hnRNP K detected in several sera of patients with SLE who commonly cDNA was modified by PCR to introduce flanking NotI and XhoI sites, develop antibodies to Ro and La proteins. To further charac- allowing replacement of the La cDNA with the hnRNP K cDNA, which terize these hY RNA-binding proteins Ro RNP complexes were resulted in an N-terminally VSV-G-tagged hnRNP K construct in reconstituted in vitro by incubating HeLa S100 extracts with pcDNA3. An N-terminally Myc-tagged version of a human hnRNP I biotinylated hY1 RNA. Reconstitution reactions employing bi- cDNA in pcDNA3 was modified to replace the Myc tag with a VSV-G tag. The integrity of the constructs was confirmed by DNA sequencing. otinylated 5 S rRNA were performed in parallel to control for As a control, the empty pcDNA3 vector was used in transfection nonspecifically binding proteins. All reactions were supple- experiments. mented with a 20-fold excess of yeast tRNA as a nonspecific African green monkey cells (COS-1) were transiently transfected competitor. Reconstituted complexes were purified by NeutrA- with the expression constructs. Briefly, COS-1 cells were grown to 80% vidin affinity chromatography followed by two-dimensional gel confluency in Dulbecco’s modified Eagle’s medium supplemented with electrophoresis and subsequently analyzed by immunoblotting 10% heat-inactivated fetal calf serum and penicillin/streptomycin in 5% CO at 37 °C. Cells were trypsinized and resuspended in phosphate- using SLE serum BM known to recognize all five novel hY buffered saline. Approximately 5 3 10 cells were transfected with 20 RNA-binding proteins as well as Ro60 and La (Ref. 28; see also mg of plasmid DNA in a total volume of 400 ml of phosphate-buffered Fig. 2A). As can be seen in Fig. 1, several protein spots on the saline. Electroporation was performed at 300 V and a capacity of 125 membrane were stained by serum BM. The spots correspond- microfarads with a Gene Pulser II (Bio-Rad). Subsequently, the cells 2 ing to proteins binding specifically to hY1 RNA are indicated by were seeded in 75-cm culture flasks and cultured overnight. arrows. The positions of Ro60 and La with its typical isoelectric Yeast Three-hybrid System—To investigate the interaction of hnRNP I and hnRNP K with hY RNAs in vivo, a three-hybrid system (36) was isoforms (37–39) were confirmed in a separate experiment us- used (RNA-protein hybrid hunter kit; Invitrogen, Groningen, The Neth- ing monospecific sera and monoclonal antibodies to these pro- erlands). The DNAs encoding hY RNAs and hY1 RNA deletion mutants teins (not shown); the two spots visible below the La spots were introduced into the SmaI and AvrII sites of the pRH59 hybrid RNA presumably represent La degradation products. Apart from the vector downstream of two copies of the MS2 RNA sequence. hnRNP I spots corresponding to Ro60 and La proteins, four major pro- and hnRNP K hybrids were constructed by ligating the PCR amplified tein spots can be detected migrating above Ro60. The most fragments into the EcoRI and XhoI sites of the pYEASTrp2 hybrid protein vector. The sequence and orientation of all recombinant DNAs acidic of these proteins, which reproducibly migrated at ;68 20714 Interaction of hnRNP I and K Proteins with hY RNAs showed pronounced binding to hY1 RNA, but only hnRNP I efficiently bound to hY3 RNA, whereas the interaction of hnRNP K with hY3 RNA was relatively weak. In this assay, neither of the two proteins interacted detectably with hY4 or hY5 RNA, confirming previous data (28). To map the regions of hY1 RNA involved in binding of the two proteins, deletion mutants of (biotinylated) hY1 RNA were used. Truncation of the Ro60 binding site did not have any effect on binding of either hnRNP I or hnRNP K (Fig. 3B, lane 5), whereas mutation of the La binding site (39-terminal UU to AG) significantly decreased binding of both proteins (Fig. 3B, lane 6). The strongest effect was observed with a mutant lack- FIG.2. Detection of hnRNP K and hnRNP I in reconstituted hY1 RNP complexes. Proteins binding in reconstitution assays to ing the pyrimidine-rich central loop 2b, which bound hnRNP I biotinylated hY1 RNA (lanes 1) or 5 S rRNA (lanes 2) were isolated by very weakly and hnRNP K not at all (Fig. 3B, lane 7). Deletion streptavidine affinity chromatography, separated by SDS-PAGE, and of stem2-loop1 or of stem3-loop3 had no or only little effect on probed by Western blotting with patient serum BM (A), a monoclonal binding of hnRNP I, whereas binding of hnRNP K appeared to anti-hnRNP K antibody (B), and a monoclonal anti-hnRNP I antibody (C). be reduced by ;50 and 80%, respectively (Fig. 3B, lanes 8 and 9). Finally, the binding of both proteins appeared to be some- kDa, was excised from a Coomassie-stained two-dimensional what increased with a mutant lacking the stem4-loop4 region gel run in parallel, digested with trypsin, and microsequenced (Fig. 3B, lane 10). A comparable result was obtained with by tandem electrospray mass spectrometry. This analysis pro- 35 S-labeled hnRNP I and K proteins translated in vitro in a vided a 19-amino acid tryptic peptide sequence (GSYGDLGG- wheat germ system (data not shown). PIITTQVTIPK), which completely matched a sequence con- Taken together, these results (i) clearly confirmed the in tained in the human hnRNP K protein (residues 378 –396). vitro binding of hnRNP I and K to hY1 RNA; (ii) showed the This result was compatible with the migration of the proteins interaction of the two proteins with hY3 RNA to be slightly in our two-dimensional gels, which largely corresponded to the (hnRNP I) or considerably (hnRNP K) weaker than with hY1 molecular mass (66 kDa) and isoelectric point values (6.1– 6.4) RNA; (iii) demonstrated the central loop 2b to be indispensable reported for hnRNP K (40). for efficient binding of both proteins to hY1 RNA; (iv) suggested The second protein that we could identify was a basic protein that La but not Ro60 is required for efficient binding; and (v) visible as a 62/60-kDa doublet. Previous experiments had al- indicated that the binding sites for the two hnRNP proteins are ready suggested the presence of a 60-kDa protein in reconsti- closely spaced but not necessarily identical. tuted hY1 RNPs that comigrated with Ro60 in SDS-PAGE and Interaction of hnRNP I and K with Native hY RNAs—To appeared to have similar RNA binding properties as the 62- investigate whether and which hY RNAs are associated with kDa protein (28). Because of its migration behavior and char- the hnRNP I and K proteins in vivo, a HeLa cell extract was acteristic doublet appearance, we hypothesized that this pro- subjected to immunoprecipitation using specific monoclonal tein was hnRNP I, the PTB, which is known to migrate as a antibodies to hnRNP I, hnRNP K, Ro60, and La. RNAs were double band of ;60 kDa in SDS-PAGE and has a reported pI of isolated from the immunoprecipitates by phenol-chloroform ex- 8.5 (40, 41). traction and probed with radiolabeled antisense hY RNAs by To confirm the presence of hnRNP I and hnRNP K in recon- Northern blot hybridization. As shown in Fig. 4A, coprecipita- stituted complexes hY1 RNA-binding proteins were separated tion of hY1 and hY3 RNA by the anti-hnRNP I monoclonal by SDS-PAGE and stained with either serum BM or mono- antibody was clearly observed although at a lower level as clonal antibodies to hnRNP K or hnRNP I, respectively (Fig. 2, compared with the precipitates obtained with the anti-La and lanes 1). Compatible with our previously published data serum anti-Ro60 antibodies, which efficiently precipitated all four hY BM recognized proteins of 80, 68, 65, 62, and 53 kDa in addition RNAs. RNA bands visible just below hY1 RNA and hY3 RNA to Ro60 and La (28). In contrast to serum BM, the anti-hnRNP (lanes 1 and 3) presumably corresponded to previously reported K antibody stained solely the 68-kDa band, whereas the anti- degradation products of these two hY RNAs known as hY2 and hnRNP I antibody recognized two bands of 62 and 60 kDa. hY3* RNAs (22, 42), which was consistent with the lack of Neither the serum nor the monoclonal antibodies recognized a coprecipitation of these molecules by the anti-La antibodies. protein in the control lanes containing proteins binding to 5 S These results not only confirmed the specific binding of hnRNP rRNA (Fig. 2, lanes 2). I to hY1 and hY3 RNA observed in vitro but also strongly Of the three other hY1 RNA-binding proteins recognized by suggested that hnRNP I is associated with hY1 and hY3 RNA the serum, the 53-kDa protein was horizontally streaked and in vivo. In contrast, coprecipitation of hY RNAs by the anti- only weakly reactive with the serum on two-dimensional im- hnRNP K antibody was not detectable (lane 4). munoblots and could therefore not be sequenced. For the 65- Although these data suggested that the association of kDa protein migrating as a double spot at neutral pI and for the hnRNP I with hY1 and hY3 RNA occurs also in vivo, they did basic protein(s) migrating at ;70 –72 kDa (presumably comi- not allow us to conclude that this protein was present also in Ro grating with hnRNP K in SDS-PAGE) no unambiguous data RNPs (i.e. hY RNA complexes containing both Ro60 and La). To could be obtained, and the 80-kDa protein was not visible on address this question, Ro and La RNPs were isolated from a Coomassie-stained two-dimensional gels. Interaction of hnRNP I and hnRNP K with hY RNAs in HeLa cell extract using anti-Ro60 and anti-La microprepara- tive immunoaffinity columns. Proteins were eluted with 1 M Vitro—To study the interaction of hnRNP I and hnRNP K with hY RNAs in more detail, reconstitution reactions were per- NaCl, separated by SDS-PAGE, and identified by immunoblot- ting using monoclonal antibodies against hnRNP I and hnRNP formed with all four hY RNAs as well as with several deletion mutants of hY1 RNA. Proteins binding to hY RNAs were sep- K; proteins isolated from in vitro reconstituted hY1 RNPs arated by SDS-PAGE, blotted onto nitrocellulose membranes, served as controls (Fig. 4B, lane 1). In these experiments the and subsequently probed with monoclonal antibodies to presence of hnRNP I in both anti-La and anti-Ro60 eluates was hnRNP I and K as described above (Fig. 3); both proteins reproducibly observed (Fig. 4B, lanes 2 and 3), demonstrating Interaction of hnRNP I and K Proteins with hY RNAs 20715 FIG.3. Interaction of hnRNP K and hnRNP I with hY RNAs and hY1 RNA deletion mutants. Reconstitution reac- tions were performed with biotinylated hY RNAs and deletion mutants of hY1 RNA, and binding of hnRNP I and K was detected by immunoblotting employing monoclonal antibodies. Note that mutant DS1L1 lacks the Ro binding site and mu- tant DS1L1sty lacks the La binding site. A, proposed secondary structure of hu- man hY1 RNA and hY1 RNA deletion mu- tants. wt, wild type; S, stem; L, loop. B, Western blot analysis of hY RNA associ- ation with hnRNP K and hnRNP I. FIG.4. Association of hnRNP I with native hY RNAs and Ro RNPs in HeLa cell extracts. A, Northern blot analysis of hY RNAs immunoprecipitated from a HeLa cell extract by monoclonal antibodies to Ro60 (lane 1), La (lane 2), hnRNP I (lane 3), and hnRNP K (lane 4). A monoclonal antibody to human interleukin-6 was used as a negative control (lane 5), and 5% of the total input RNA served as a positive control (lane 6). B, Western blot analysis of hnRNP I immunoprecipitated by the anti-La (lane 2) and anti-Ro60 (lane 3) monoclonal antibodies; a monoclonal anti-interleukin-6 antibody served again as a negative control (lane 4). For comparison hY1 RNA-associated proteins isolated from in vitro reconstituted complexes are shown (lane 1). that this protein was contained in Ro RNP complexes. On the Yeast Three-hybrid System—To investigate the interactions of other hand, and in agreement with the RNA precipitation data, hnRNP I and hnRNP K with hY RNAs in a living cell, we made hnRNP K could not be detected in these eluates (not shown). use of the yeast three-hybrid system (36). In analogy to the To confirm these data and to account for the possibility that widely used two-hybrid system, this system is based on tran- the epitope recognized by the anti-hnRNP K monoclonal anti- scriptional activation of the reporter genes his3 and lacZ upon body might be inaccessible when the hnRNP K is complexed interaction of RNA with the protein of interest. In our case the with hY RNA, we expressed both hnRNP proteins and, as a first (protein) hybrid consisted of the DNA-binding domain of control, La as VSV-G-tagged fusion proteins in transiently the transcriptional activator LexA fused to the RNA-binding transfected COS-1 cells. Western blot analysis with a mono- viral MS2 coat protein (LexA DBD-MS2), the second (RNA) clonal antibody directed to the VSV-G tag demonstrated that hybrid consisted of hY RNA (or hY1 RNA deletion mutants) the three tagged proteins are expressed to similar amounts cloned downstream of the MS2 RNA sequence (MS2 RNA-hY (Fig. 5A). This antibody was then used to immunoprecipitate RNA), and the third (protein) hybrid was composed of the lysates of transfected cells that were subsequently analyzed for transcription activation domain of Gal4 fused to either hnRNP the presence of (coprecipitated) Y RNAs by Northern blot hy- I or hnRNP K (B42AD-hnRNP protein) (Fig. 6A). bridization. Also by this approach hnRNP I was found to asso- To examine the interaction of hY1 and hY3 RNA with ciate with Y1 and Y3 RNA (Fig. 5B, lane 5), and again no hnRNP I or hnRNP K, a yeast strain expressing the LexA association of hnRNP K with Y RNAs could be detected (Fig. DBD-MS2 coat protein hybrid was cotransformed with the 5B, lane 8). As expected, VSV-G-tagged La was associated with hybrid plasmids encoding these RNAs and proteins. Transfor- all four Y RNAs (lane 2), and no Y RNAs were detectable when mants lacking any of the (RNA or protein) hybrid components cells were transfected with an “empty vector” (lane 11). All four were not able to grow on a medium deficient of histidine (not Y RNAs were also coprecipitated by the anti-La (lane 3) and shown) and showed no or only little b-gal activity when grown anti-Ro60 (lanes 6, 9, and 12) antibodies. on a synthetic medium lacking uracil and tryptophan (Fig. 6B). Interaction of hnRNP I and K with hY1 and hY3 RNA in a In contrast, elevated b-gal activity was clearly seen in trans- 20716 Interaction of hnRNP I and K Proteins with hY RNAs this has not been presented yet (3, 4). Here we have identified two of the proteins previously described by us to associate with hY RNAs in vitro (28) as hnRNP I and hnRNP K. The two proteins interacted strongly with hY1 RNA and to a lesser extent with hY3 RNA but not with hY4 or hY5 RNA, which is in agreement with our previous findings. Immunoprecipitation experiments performed with extracts from HeLa cells and tran- siently transfected COS-1 cells provided substantial evidence for in vivo association of hnRNP I with hY1 and hY3 RNA and indicated the existence of a subpopulation of Ro RNPs contain- ing hnRNP I in addition to Ro60 and La. The observation that the anti-hnRNP I antibody precipitated significantly smaller amounts of hY RNAs than the anti-Ro60 antibody (although comparable amounts of Ro60 and hnRNP I appeared to be bound in the reconstitution assays) indicates that, in contrast to Ro60, hnRNP I is associated with a minor portion (10 –20%) of hY1 and hY3 RNAs. Although these assays did not provide any evidence for hY RNA-hnRNP K interactions in these cel- lular extracts, a clearly positive result was obtained in a yeast three-hybrid system that was comparable with that obtained with hnRNP I. Thus, hnRNP K may bind only to a rather minor subset of hY RNAs that are devoid of Ro60 (there is no Ro60 homologue in yeast) and therefore difficult to detect, or, alter- natively, the interaction with hY RNAs may have been dis- turbed during preparation of the cellular extracts or upon antibody binding in immunoprecipitation assays. The importance of an intact La binding site for efficient binding of hnRNP I and hnRNP K was remarkable and leads us FIG.5. Association of VSV-tagged hnRNP I with Y RNAs in to speculate that the function of La in hY RNP assembly may transiently transfected cells. COS-1 cells were transfected with constructs encoding VSV-G-tagged La, hnRNP I, and hnRNP K, and an be that of an RNA chaperone being required for correct folding empty vector (mock), and 24 h after transfection cell lysates were of hY RNAs, thus enabling binding of other proteins (with the prepared and analyzed by Western blotting (A) or Northern blotting (B). notable exception of Ro60). This would be consistent with re- A, expression of the VSV-G-tagged proteins La, hnRNP I, and hnRNP K analyzed by Western blotting using a monoclonal anti-VSV-G tag anti- cent reports on the chaperoning role of La in small nuclear body. The positions of the molecular mass markers are indicated on the RNP assembly and pre-tRNA processing (12, 15, 19). right. B, Northern blot analysis of Y RNAs coprecipitating with VSV- The results obtained with hY1 RNA deletion mutants G-tagged proteins. The lysates were subjected to immunoprecipitation showed that the internal pyrimidine loop (71– 86 nucleotides) with monoclonal anti-VSV-G tag, anti-La, or anti-Ro60 antibodies, re- was indispensable for efficient association of the two hnRNP spectively. RNA isolated from the total lysates (input) and from the immunoprecipitates was analyzed by Northern blot hybridization using proteins with hY1 RNA and demonstrated their binding sites to antisense hY RNAs as probes. The positions of Y1, Y3, Y4, and Y5 are be clearly different from the Ro60 binding site. Both hnRNP I indicated. The band indicated with ** probably represents a degrada- and K are known to bind to pyrimidine-rich sequences (43, 44). tion product of Y3, previously designated Y3** (42). Thus, hnRNP I, which is commonly known as PTB, binds to the polypyrimidine stretch present near the 39 splice site of many formants expressing hY1 RNA and hnRNP I or hnRNP K introns and also to pyrimidine-rich sequences of mature hybrids, respectively, whereas b-gal activity exceeded back- mRNAs (45). For hnRNP K, which shows increased affinity for ground levels only by approximately 2-fold in hY3 RNA trans- poly(rC) sequences but does not bind to the polypyrimidine formants (Fig. 6B). The observed interactions of hY1 RNA with tract, several cytosine-rich recognition motives have been de- the two hnRNP proteins in the yeast system were largely scribed, such as the CT element (CCCTCCCCA) of the c-myc consistent with the in vitro reconstitution data described gene (46) and CU repeats (CCCCACCCUCUUCCCC) present above. Interactions of hnRNP I with hY3 RNA, on the other in the 39-untranslated region of erythroid 15-lipoxygenase hand, were considerably weaker than in the in vitro binding mRNA (47). These sequences as well as those recognized by experiments. hnRNP I (PTB) show significant similarities with the central When we examined binding of hnRNP I and K to hY1 RNA loop 2b region of hY1 RNA (UACUCUUUCCCCCCUU), sup- deletion mutants (same as shown in Fig. 3), mutant Y1DL2b porting the assumption that this region may directly interact lacking the pyrimidine-rich domain showed only low levels of with both proteins as suggested by the results obtained with b-gal activity when cotransformed with either hnRNP K or the deletion mutants. Remarkably, an internal pyrimidine-rich hnRNP I, again compatible with the in vitro results. In con- loop is present also in hY3 RNA, whereas it is lacking in hY4 trast, b-gal activity of the Y1DS3L3 mutant strain was even and hY5 RNA, which would be consistent with their failure to somewhat greater than that of wild-type Y1 RNA for both bind the two hnRNP proteins. hnRNP I and K, whereas deletion of stem4-loop4 (Y1DS4L4) The heterogeneous nature of Ro RNPs has been postulated led to a slightly decreased activation of the lacZ gene with both by several researchers based on biochemical fractionation and hnRNP proteins. The other two hY1 RNA mutants showed in vitro reconstitution data (22, 23, 28). The number of Y RNAs similar b-gal activities as wild-type hY1 RNA. has increased throughout the evolution from C. elegans, which DISCUSSION expresses only one Y RNA, to vertebrates with four Y RNAs (6, Although data from several laboratories suggest that Ro 48). Interestingly, the Ro60 binding site and the pyrimidine- RNP complexes may contain a number of other proteins in rich loop sequence represent the most conserved regions, being addition to Ro60 and La (20 –28), unambiguous evidence for already present in C. elegans RNA, which shows the greatest Interaction of hnRNP I and K Proteins with hY RNAs 20717 FIG.6. Interaction of hnRNP I and hnRNP K with hY1 and hY3 RNA in a yeast three-hybrid system. The yeast three-hybrid system was used to study interaction of the two hnRNP proteins with hY RNAs in vivo by measuring b-gal activity of transformed cells. A, schematic diagram showing gene activation by spe- cific interaction between hY-RNA and hnRNP I or K proteins in the yeast three- hybrid system. Expression of reporter genes lacZ and his3 is activated upon pos- itive interaction between hnRNP I or K and hY1 RNA dragging the following two domains of the transcriptional activator to a close vicinity: the DNA-binding do- main of LexA (DBD) and the activation domain of Gal 4 (AD). B, analysis of b-gal activity. Transformants were grown on synthetic medium lacking uracil and tryptophan, and b-gal activity was tested in the blue color X-gal filter assay (right) and quantitatively determined by direct measurement of the enzymatic activity in units/mg yeast mass (left). homology to hY3 RNA (49). This strongly suggests that the shown to bind specifically to several viral internal ribosome pyrimidine-rich element is important for Y RNA functioning, entry sites, thereby promoting ribosome binding in a cap-inde- including interactions with other proteins and/or RNAs. Fur- pendent manner (62– 64). Remarkably, a similar activity was thermore, the internal loop of hY1 RNA was shown to be also found for La (13), and cooperation of these two proteins in resistant to enzymatic and chemical cleavage (50), an observa- the regulation of internal ribosome entry site-dependent trans- tion also made with pyrimidine-rich loops of other small RNAs lation has been recently suggested (65). Furthermore, these transcribed by RNA polymerase III (51–53). Because the four proteins may be also required for efficient and correct initiation vertebrate Y RNAs show a high diversity in the central region, of cap-dependent translation by inhibiting translation of un- this part of the molecule might determine the fate (i.e. local- capped mRNAs (66). Apart from its role as transcription factor ization and function) of Y RNPs or Ro RNPs, respectively, (44, 67, 68), hnRNP K protein has been demonstrated to act as because of differential association with proteins of (more or a differentiation regulator by virtue of its binding to the control less) diverse function (such as hnRNP I and hnRNP K). elements at the 39 end of the erythroid 15-lipoxygenase mRNA, Several RNA polymerase III transcripts including RNase P thereby inhibiting translation (47). In a similar manner trans- RNA, RNase MRP RNA, and hY RNAs (except hY4 RNA) have lation of papilloma virus late mRNAs is inhibited by hnRNP K been localized to the perinucleolar compartments together with and the poly(rC)-binding protein (69). Furthermore, two trans- two hnRNP proteins, hnRNP I (41, 54), and CUG-binding pro- lation-related functions have been assigned to the Ro60 pro- tein/hNab50 (55, 56). Importantly, the presence of hnRNP I in tein, namely participation in a degradation pathway for mis- the perinucleolar compartments was found to be sensitive to folded 5 S rRNA (17) and a role in the regulation of translation RNase A treatment (57), which may be considered a further of ribosomal protein L4 in concert with La and cellular nucleic (though rather indirect) indication for in vivo interactions of acid-binding protein by binding to a polypyrimidine stretch in this protein with hY1 and hY3 RNAs. Remarkably, the pres- the 59-untranslated region of L4 mRNA (16, 70). ence of Ro60 and La could not be detected within the pe- Combining our experimental data and those of others one rinucleolar compartments (54), which may suggest the existence may speculate about the cellular role of Ro RNPs or hY RNPs, of hY RNA complexes containing hnRNP I (as well as other respectively: newly transcribed hY RNAs associate with La and proteins yet to be identified) but devoid of both Ro60 and La. move through the nucleoplasm toward the nuclear membrane hnRNP I and K are both shuttling proteins that belong to a where Ro60 associates just prior to export into the cytoplasm, group of multifunctional RNA-binding proteins exerting regu- being indispensable for nuclear export of hY RNAs (3, 9). Dur- latory roles at the post-transcriptional level, including RNA ing their intranuclear migration subsets of hY RNPs are processing and export as well as regulation of mRNA stability formed by virtue of association with various proteins including and translation (44, 58 – 61). Given that Ro RNPs are predom- hnRNP I and K, Ro52, and presumably other proteins includ- inantly, if not exclusively, localized in the cytoplasm, it is ing those reported to interact with hY RNA in vitro or with important to mention reported functions of hnRNP I and K that Ro60 in yeast two- and three-hybrid systems (26 –28). All these are related to translational events. Thus, hnRNP I has been proteins may be multifunctional and upon binding to hY RNAs 20718 Interaction of hnRNP I and K Proteins with hY RNAs 28. Fabini, G., Rutjes, S. A., Zimmermann, C., Pruijn, G. J. M., and Steiner, G. become destined to exert specific functions either in the nu- (2000) Eur. J. 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