Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

The first and third uORFs in RSV leader RNA are efficiently translated: implications for translational regulation and viral RNA packaging

The first and third uORFs in RSV leader RNA are efficiently translated: implications for... Downloaded from https://academic.oup.com/nar/article-abstract/23/5/861/2376433 by DeepDyve user on 06 August 2020 11995 Oxford University Press Nucleic Acids Research, 1995, Vol. 23, No. 5 861-868 The first and third uORFs in RSV leader RNA are efficiently translated: implications for translational regulation and viral RNA packaging Olivier Donz6*, Pascal Damay and Pierre-Francois Spahr Department of Molecular Biology, University of Geneva, 30 Quai E. Ansermet, 1211 Geneva 4, Switzerland Received September 1,1994; Revised and Accepted January 24,1995 ABSTRACT These three elements are situated at different strategic places within the leader. The first and second uORFs are found within Rous sarcoma virus (RSV) RNA leader contains three the proximal 5' secondary structure-rich region while the third short upstream open reading frames. We have shown uORF is located close to the packaging signal (termed y) , which recently that both uORFs 1 and 3 Influence in vivo is required for efficient recognition of the viral RNA by translation of the downstream gag gene and are /ra/u-acting factors (8-10). Previous studies have shown that involved in the virus RNA packaging process. In this AUG1 is the main ribosome binding site in the RSV leader report, we have studied the translational events (11-13) and that the encoded heptapeptide product is synthesized occurlng at the upstream AUGs in vivo. We show that in vitro (14). Furthermore, mutations that alter the RSV leader (i) the first and third AUGs are efficient translational AUGs increased downstream translation in vitro only slightly initiation sites; (II) ribosomes reinitiate efficiently at (15), yet have profound effects on viral replication (16—18). In a AUG3; and (Hi) deletions in the intercistronlc distance recent study, we characterized the role of the three uORFs present between uORF1 and 3 (which is well conserved among in the leader of Rous sarcoma virus (Prague C strain). We reported avian strains) prevent ribosome Initiation at AUG3, that uORFl and 3 are key elements involved in the viral life cycle thus Increasing translation efficiency at the down- and act by regulating the efficiency of translation at the stream AUGgag. The roles of the uORFs in translation downstream AUGgag as well as the efficiency of viral RNA and packaging are discussed. packaging (18). Mutation of either AUG1 or 3 has a profound effect on RNA encapsidation, inhibiting this process by INTRODUCTION 20-50-fold. We proposed a model whereby ribosomes first translate uORFl and then subsequently reinitiate and pause at the In eukaryotic cells, translation is usually initiated in a manner AUG3. We postulated that initiation at the third AUG is the consistent with the ribosome scanning model (1). According to central step in the regulation of RNA packaging. Reinitiation at this model, the 40S ribosomal subunit and translation initiation AUG3 would impede temporarily the flow of ribosomes on the factors bind to the 5 ' end of mRNA and scan the RNA leader until RSV leader, clearing the \\f packaging sequence present in the they reach an AUG codon in the appropriate context. Subsequent- leader just downstream of uORF3 (18). ly, the 60S ribosome joins and initiates polypeptide chain In the present study, we examined this prediction of our elongation (2). The scanning model accounts for the effects of proposed model in vivo. To this end, we looked at the translation structural features within the 5' untranslated region, such as initiation rates at each AUG present within the RSV leader RNA. secondary structure and open reading frames, which can strongly The reporter gene firefly luciferase was fused to all constructs to influence the efficiency of translation initiation. The presence of allow quantitation of translational initiation rates at the different one or more upstream open reading frames (uORFs) has been AUGs. The reinitiation hypothesis between uORFl and 3 was shown to influence the translation of downstream ORFs. also tested by altering the intercistronic distance. Our data reveal Initiation occurs preferentially at the upstream site, which in turn that uORFl and 3 are efficient initiation sites for translation and reduces initiation downstream due to the apparent inefficiency of strongly suggest that AUG3 is recognized by reinitiating reinitiation at internal AUG codons. Therefore, in most cases, ribosomes. We also showed the effects of these RSV RNA leader uORFs inhibit downstream translation in proportion to the alterations on the efficiency of viral RNA packaging. efficiency of their own translation (1). In a few cases, the coding capacity of the uORF was found to influence the downstream translation (3-6). MATERIALS AND METHODS In Rous sarcoma virus, the 5 ' leader contains three short open Cell culture reading frames of 7, 16 and 9 codons in length. The three uORFs are conserved in length, and in the nucleotide sequence surround- Chicken embryo fibroblasts prepared from Spafas eggs (Gs~ and ing the initiation codons among the avian/leukosis viruses (7). Chf~: Norwich, CT, USA) were grown in Dulbecco-modified *To whom correspondence should be addressed at present address: Department of Biochemistry, McGill University, 3655 Drummond Street, Montreal, Quebec H3G 1Y6, Canada Downloaded from https://academic.oup.com/nar/article-abstract/23/5/861/2376433 by DeepDyve user on 06 August 2020 862 Nucleic Acids Research, 1995, Vol. 23, No. 5 Eagle medium containing 5% fetal calf serum (Gibco Labora- performed the same scheme to construct InsDel from BsDel44. tories, Grand Island, NY) at 41 °C in an atmosphere supplem- The same pSp73 insert was used and again a double insert was ented with 5% CO2. present in the construct, but in the same orientation. InsDel carries, thus, a 94 nt insert at position 102 in addition of the 44 nt deletion: the intercistronic distance in this construct is 184 nt Bacterial strains instead of 134 nt. In Del58, we removed the 58 nt RsrU-BstEll Escherichia coli DH5cc and CJ236 were grown according to the fragment (positions 47 and 102, respectively). Del 13 was instructions of the mutagenesis kit (Biorad). E.coli DH5a was constructed by digesting the Bslead clone with BstEll followed rendered competent as previously described (19). Plasmid DNAs by treatment with mung Bean nuclease. We obtained a 13 nt were purified from either small or large bacterial cultures by the deletion from nucleotides 96 to 108. The introduction of the alkaline lysis method and for transfection were further purified by mutations was confirmed by the dideoxy-chain termination precipitation with polyethylene glycol (PEG) (19). method of DNA sequencing using T7 polymerase (Pharmacia) primed by a synthetic oligonucleotide complementary to the 3' Cloned DNAs end of the RSV leader. The mutated fragments were cloned back into pAsPrc using the Sphl sites and then the SaH-EcoRN Plasmid pAPrc has already been described (20); it contains a fragment was introduced into pAPrc (20). non-permutated copy of the provirus RSV Prague C strain. Plasmid pAsPrc is a Sall-EcoRV subclone of pAPrc in pBR322 Transfection containing the entire leader and gag sequences. All the mutations were constructed in Bslead, a 1167 bp Sphl fragment of pAsPrc Cells either freshly prepared from embryos or frozen in the cloned in the phagemid vector Bluescribe (-) (Stratagene, San presence of glycerol were used for transfection after two to seven Diego, USA). passages. Cells were transfected using the DEAE-Dextran To construct the plasmid carrying the luciferase gene (Bsluc), procedure as described previously (18). the Bsml-BamUl fragment (with the ends filled by the Klenow fragment of DNA polymerase I) from pRSVluc (21) containing Protein analysis the luciferase gene was cloned by blunt end ligation into Viral particles produced by the transfected cells were purified by BsleadMin to replace the HindUl fragment encoding the gag gene ultracentrifugation through a 20% sucrose cushion and their (the HindJQ ends were similarly filled by Klenow prior to protein content analyzed by immunoblotting with polyclonal ligation). BsleadMin carries the 1167 bp Sphl fragment of pAsPrc antibodies against RSV CA (p27), as described previously (20). in which the AUG initiator of the gag gene at position 380 has been mutated to TTC in order to create a HindUl site at the Luciferase assay beginning of the gag gene (22). Bsluc contains the RSV leader fused to the luciferase gene, including 20 nucleotides (nt) of the Each 10 cm plate of transfected CEF was washed three times in luciferase leader. A 440 nt Pstl fragment from each Bslead mutant phosphate-buffered saline without Ca2+ and the cells were was cloned into the corresponding site of Bsluc to replace the harvested in 500 (il of lysis buffer (Promega). Cell debris was sequence derived from BsleadMin. pelleted by centrifugation in a microcentrifuge for 5 min at 4°C. A 20 \i\ aliquot of extract was added to 100 u.1 of assay buffer Construction of mutants in RSV leader (Promega) in a small test tube. Luciferase activity was measured in millivolts in a luminometer (Bioorbit) using the luciferase The following oligonucleotides were synthesised on an Applied assay system (Promega). Biosystems 381 A DNA synthetizer and purified as previously Total luciferase RNA was isolated from transfected CEF using described (23): the guanidinium/CsCl method (19). RNA was then digested with DNase I and subjected to a RNase ONE protection (Promega) M3189: 3'-GCAGAGCGAATAAGCrCCTACCCrGCAGTTGGGAT- because RNA levels were too low for decisive Northern CATCTCCCCC-5' experiments. Prior to conducting the experiments shown.in SEL3:3'-CTCCCCCGACGCCGAAATCCTCCCGTCTTC-5' Figures 2 and 5, we evaluated this 'RNase One' by using different Del44:3'-ATCAATCCCTTATCACCAAAGCCCCTCGCC-5'par amounts of pure luciferase RNA (from Promega). The data from ORFl-luc: 3'-AACTACCGGCCTGGCAGCTAAGGGCTTCTGCG- this experiment convinced us that this enzyme gave a linear and GTTTTTGTAT-5' quantitative signal, although residual undigested probe was still ORF2-luc: 3'-CTGGGGCTGCACTATCMTCCCTTCTGCGGTnT- present. To obtain protected fragment of the same size for TGTATTTC-5' luciferase constructs, a 413 RNA antisense probe was synthesized ORF3-luc: 3'-TGGGATCATCTCCCCCGACGCCGACrTCTGCGG- from the pGEM-luc (Promega) digested with £coRV; this probe TTTTTGTATTTC-5' contains homologies to the last 356 nt of the luciferase ORF. AUG31uc: 3'-GCGAATAAGCCCCTCGCCTGCTACCTTCTGCGG- TTTTTGTATTTCTTTCCG-5' Purification of viral RNA AH the mutants cited above were constructed as described previously (24). The viral RNA of virions produced after transfection was purified The mutant Ins was made by digesting the Bslead clone (20) and analyzed by RNase protection assay. The RNA was extracted from virions as described previously( 18). Total cellular RNA was with BstEU (position 102). A BglU-SaCl insert (from plasmid purified from subconfluent cultures by lysis in guanidinium pSp73: Promega) was treated with mung bean nuclease and ligated to Bslead to give pAsIns: two inserts in inverted thiocyanate, followed by centrifugation through cesium chloride orientation are present, adding 94 nt to the RSV leader. We as described (19). Downloaded from https://academic.oup.com/nar/article-abstract/23/5/861/2376433 by DeepDyve user on 06 August 2020 Nucleic Acids Research, 1995, Vol. 23, No. 5 863 own Bs WT luciferase Bs ORF1 luciferase . Bs ORF2 tuciferase , 0RF2 0RF3 tucfftrss* Bs ORF3 luciferase , 7777X AUO 0RF1 0RF2 AU63 \ixH&r%%m Bs AUG3 luciferaie, 1 i i Relative Ljdferase activity Figure 1. Efficiency of translation initiation at the different AUGs present in RSV leader. The schematic depicts the 5' end encoded by the different luciferase fusion construct (drawn to scale). The uORFs are represented by boxes. The hatched boxes symbolize the uORFs involved in translation and packaging rcgulation (9). The numbers indicate the first AUG triplet for each uORF-luciferase fusion and the AUG initiator of the luciferase gene. Levels of luciferase activity are shown at the right and arc given relative to the control plasmid with the whole RSV leader (BsWT-luciferase); values have been normalized to tbe RNA levels (Fig. 2). The light units produced by any given luciferase expression vector varied by <10% in parallel transfections using two independent clones for each construct The emitted light was quantitated in a Bioorbit luminometer. Quantitation of RNA present in the virions or in the cell were luciferase activities observed were due to differential transla- performed using an RNase-protection assay. Plasmid pL(-), tional efficiencies rather than transcriptional variations, we which contains the EcoRl-Xhol fragment from pAPrC (20), was monitored levels of luciferase RNA within the transfected cells by digested with Sad and in vitro transcribed using T7 polymerase RNase protection analysis. A representative protection experi- and a commercial kit (Promega) according to the kit instructions. ment is shown in Figure 2, using a probe which protected 356 nt The antisense RNA probe contains homologies to 355 nt of the of the luciferase mRNA. In several experiments, we observed leader and the gag gene. Plasmid pL(-) was also digested with similar levels of RNA synthesized from each construct, indicating BstEU and in vitro transcribed using T7 polymerase. The that the variations in luciferase activity truly reflected altered antisense transcript produced was used to detect the presence of translational efficiencies of the recombinant RNAs. the mutations in viral RNA extracted from the different mutant The luciferase activities in extracts from cells transfected with virions (data not shown). RNase protection was performed with each construct were normalized to the activity in cells transfected RNase One (Promega) as described by the manufacturer. The with BsWT-luciferase construct, which contained the whole RSV nuclease resistant hybrid was analysed on a denaturing polyacryl- leader fused to the luciferase sequence (Fig. 1 and ref. 18). amide gel and the product detected by autoradiography. Translational efficiency at the first AUG was measured in the BsORFl-luciferase plasmid which produced as much as 4-fold RESULTS more activity than at the AUGgag (compare Bswtluc with BsORFlluc in Fig. 1). The luciferase activity present in the lysate Upstream AUG codons 1 and 3 in RSV leader RNA are of cells transfected with BsORF2-luciferase was no more than 3% efficient initiation sites of the activity observed at the AUGgag and even less if we compare to the efficiency at AUG1. This indicates that AUG2 is We showed previously that alterations of AUG 1 or AUG3 present poorly recognized by ribosomes during the scanning process, in in the RSV leader RNA influences translation of the gag gene, agreement with previous observations (18,29). To investigate situated downstream of the uORFs. uORFl is a translational initiation occuring at AUG3, we used two different fusion enhancer of viral proteins, while uORF3 inhibits downstream plasmids (Fig. 1): in the BsORF3-luciferase construct, the translation at the AUGgag (18). These results strongly suggested luciferase was fused to the end of the third reading frame, whereas that ribosomes initiate and translate both uORFl and uORF3 in in the BsAUG3-luciferase plasmid, the reporter sequence was vivo. To study further the translational events occuring at the RSV directly fused to AUG3. Both constructs showed that AUG3 is an leader uORFs, we fused the end of each uORF to the coding efficient initiation codon (2-fold increase compared to AUGgag sequence of the firefly luciferase gene (21). Fusion constructs in Bswtluc; Fig. 1). Although a precise comparison of the levels have been successfully used for a better understanding of the uORFs present in the leader of the GCN4 gene in the yeast of expression of the fusion proteins could not be made without Saccharomyces cerevisiae (25). Since the luciferase coding knowledge of differences in the stability and specific activity of region was fused at the end of the uORFs in order to minimize each, the results support the idea that both uORFl and uORF3 are artifacts due to the fused gene, we reasoned that expression of the sites for efficient translation initiation. uORF-luciferase fusions should reflect the translational rates of To rule out the possibility that luciferase activities observed unmodified uORFs (see Discussion). For each construct, we used were due to recognition of a cryptic initiation codon present two independent clones. The expression from each fusion within the luciferase coding region (21) rather than those within construct was tested by transient transfection into chicken the RSV leader region, we mutated the initiation AUG into a embryo fibroblasts (CEFs). To ensure that differences in non-initiation codon for each uORF fusion construct. The control Downloaded from https://academic.oup.com/nar/article-abstract/23/5/861/2376433 by DeepDyve user on 06 August 2020 864 Nucleic Acids Research, 1995, Vol. 23, No. 5 AUG codon (26-28). This was ascertained by studying the luciferase gene fused to the RSV leader. The Sel3 mutation did not affect the mRNA level as observed by RNase protection analysis (Fig. 2). As expected, the lengthened version of uORF3 affected the translational efficiency at AVGgag, causing a 3-fold decrease in luciferase activity compared to wild-type value (35% -* - Probe translation efficiency compared to WT) (Fig. 3). Together with -* - Protected results of uORF-luciferase fusion studies, these data point out the fragment strong initiation potential of the third AUG, contradicting a previous study which showed indirectly that AUG3 is initiated to a small extent (29). Insertion of a sequence with the potential to form stable secondary structures greatly inhibits protein expression initiated at AVGgag The presence of a sequence with the potential to form stable secondary structures in the 5' mRNA leader generally inhibits translation in eukaryotes, presumably because it interferes with the scanning process (30-32). In the case of poliovirus mRNA, — - Probe evidence was presented that these negative elements are without —•- Protected effect as the mRNA is initiated by the process of internal initiation fragment (33). To determine whether ribosomes can overcome such an inhibitory element in the RSV leader, we introduced a sequence capable of forming a stable secondary structure between uORFl and uORF3 (mutant 'Ins' Fig. 3). This sequence was extremely stable (>150 Kcal/mol) and was formed by a 94 nt insert with dyad symmetry. When transcribed into mRNA, it is predicted to form a stable hairpin structure. We introduced this sequence at S'c*p position 102 within the RSV leader which is 38 nt downstream EcoH Ludferai e mRNA IUO from the uORFl termination codon and 96 nt upstream from the 4l3nt -* - Probe uORFi initiation site. Sequences with similar predicted second- ary structure have been shown to reduce greatly cap-dependent Protected fragment translation in eukaryotic cells (30,32). As shown in Figure 3 (compare WT with Ins), the presence of this stable structure within the leader strongly inhibits translation initiation at the Figure 2. RNase protection analysis of luciferase containing mRNAs produced by the different Bsluciferase constructs. On the top is the analysis of protected AVGgag (>1% of WT). The observed defect was manifested at fragments by electrophorcsis in a polyacrylamide sequencing gel. Bands were the level of translation, since analysis of mRNA showed similar visualized by autoradiography. Names of the different constructs designed in levels of transcript (Fig. 2). To test whether this insertion was Figures I and 3 are shown above the corresponding lane. Undigested probes inhibitory due to the stem structure or simply because of its and protected fragments are indicated on the right. At the bottom is shown a schematic illustration of the probe used in this experiment The £coRV particular sequence, we inserted the same sequence without dyad represents the 3' end of the RNA probe. symetry in the same location (see Materials and Methods and Fig. 3: InsDel). The presence of this unstructured sequence does not affect translational efficiency at the AVGgag. This indicates that constructs were transfected into CEFs and luciferase activity was secondary structure in the Ins construct was responsible for.the monitored in the lysate of transfected cells. None of the cells observed effect, rather than the sequence itself (compare Ins and transfected with control plasmids produced any detectable InsDel: Figs 3 and 4). These data, taken together with the luminescer.ee and as such were comparable to the luciferase observations reported above, strongly suggest that translation of activity from lysates of the mock-transfected cells (data not uORFs and of the gag gene occurs in vivo by a ribosome scanning shown). This demonstrated that the luciferase data reported above along the RSV leader. These data also fit with previous results reflect the use of the RSV leader AUGs. showing that in vitro a stable secondary structure present within the RSV leader decrease translation to 10% compared to the WT Elongation of uORF3 diminished translation at the level (15). AVGgag To investigate whether the initiation potential of AUG3 in the Effect of intercistronic length between uORFl and 3 on full-length virus leader parallels that of the luciferase-fusion downstream translation construct, we lengthened uORF3 in the RSV leader by addition of a single base (a thymidine) at the end of uORF3 (Sel3: Fig. 3). Efficient reinitiation is dependent on intercistronic length. It has This addition shifts the frame of the third uORF, leading to been observed that inhibition of preproinsulin synthesis by an termination 11 codons downstream of the AVGgag. Since uORF increased as the intercistronic distance was decreased from uORF3 in Sel3 overlaps the gag sequence, translation initiation 79 to 2 nt (34). In our system, the uORFl and 3 are separated by at AUG3 codon is expected to interfere with initiation at the gag 134 nt and this interval is well conserved between the different Downloaded from https://academic.oup.com/nar/article-abstract/23/5/861/2376433 by DeepDyve user on 06 August 2020 Nucleic Acids Research, 1995, Vol. 23, No. 5 865 WT Sel 3 ORFJ V//A M31" Aue Vc* OBF3 AUSn aian *U0« Ael58 0BF3 AUS H V///A Ael13 ORF3 V///A InsAel 0KF3 lom Y///A Ins 0 1 2 3 Relative Uidferase activity Figure 3. Translational efficiency of the mutants in the RSV leader. The scheme shows the different mutants with alterations in the intercistronic distance between uORFl and uORF3 as well as in uORFl and uORF3. The functional uORFs are represented in hatched boxes. Levels of luciferase activity are given relative to the control plasmid with the whole RSV leader (BsWT-luciferase). The light units produced by any given luciferase expression vector varied by <1O% in parallel transfections (see Materials and Methods). the surrounding sequences are dispensable for regulation of both translation and packaging (36,37). Restriction sites present within the first (RsrQ) and second uORFs (BstEU) were used to delete 58 nt and to create a new uORF beginning at AUG1 and terminating at the uORF2 stop codon. This mutant (Del58) possesses an uORFl of 12 codons terminating at position 130, thus situated at 68 nt upstream of the third initiation codon. For mutant M3' 89 , AUG 3 was mutated to UCA (as in pAM3:18) and CA— a new AUG (in a good context for translation: AGGATGG) was created at position 189 to replace an AGC codon. In the latter case, translation of the new uORF3 would occur in a - 1 frame producing a pentapeptide product. Moreover, the intercistronic interval was decreased by 11 nt in this mutant The mutants Del44 and Del 13 did carry intact uORFl and 3, while the two other mutants, Del58 and M3189 have a modification in uORFl or Figure 4 . Analysis of the virion gag-encoded proteins. Virions produced by the uORF3, respectively. transfected cells were purified as described in Materials and Methods. Viral To evaluate the effect of these deletions on translation initiation proteins were resolved by SDS-PAGE and immunoblotted with polyclonal at the AUGgag codon in vivo, we measured the emission of light antibodies against RSV CA (p27) and detected with 123I-labelled protein A. produced by the luciferase fusion constructs under the control of The mutants designated in Figure 3 are indicated above each lane. Lane C indicates the control cells, mock transfected with no DNA. the AUGgag codon (Fig. 3). For each construct, two independent clones were used, giving identical results. Moreover, in several experiments, we showed that mRNAs of the mutants were avian strains (7,35). If reinitiation occurs in the RSV leader, then transcribed to similar levels (Fig. 2). decreasing the distance between uORFl and uORF3 should allow The 44 nt deletion (Del44) led to a 2-fold increase in ribosomes to bypass the uORF3, thus increasing translation translational efficiency at the downstream AUG gag codon. The efficiency downstream at the AUGgag, as observed in the enhanced translation produced by this deletion mimics the effect absence of AUG3 (18). To test this possibility, several mutants caused by mutation of AUG3 (2-fold increase; 18). One possible with decreasing distance between both uORFs were made and interpretation of this result is that the intercistronic interval is too two independent clones were used for each mutant (Fig. 3). The short to allow efficient reinitiation at AUG3 (34), thus permitting first construct (Del44) has a 44 nt deletion between the the ribosomes to initiate downstream at the AUGgag more minicistrons (positions 137-181). Another carries a 13 nt deletion efficiently. More surprising was the result obtained for Del 13: within the uORF2 (Del 13); we chose to delete this region because shortening of the intercistronic space between uORFl and 3 by Downloaded from https://academic.oup.com/nar/article-abstract/23/5/861/2376433 by DeepDyve user on 06 August 2020 866 Nucleic Acids Research, 1995, Vol. 23, No. 5 allow ribosomes to initiate at AUG3, thereby inhibiting transla- tion initiation at AUGgag. To test this prediction, we inserted 94 nt into Del44 between uORFl and uORF3 at a place devoid of assigned function (position 102; see InsDel: Fig. 3). Thus, this leader carries a 94 nt insertion coupled with a 44 nt deletion, to rebuild an intercistronic distance greater than the wt distance. The new hybrid intercistronic interval restored completely the rein- • *— •+— Probe itiation competence of the ribosomes to wild-type level (Fig. 3), revealing that a longer distance between uORFl and uORF3 does - Protected not favor more efficient reinitiation. Accordingly, 134 nt appears fragment to be the preferred reinitiation distance for the virus: increasing it does not enhance reinitiation efficiency, whereas deletion of only 13 nt strongly impairs reinitiation. The experiments presented here are consistent with the idea that reinitiation is distance-dependent and strongly suggest that reinitiation is involved in RSV translational regulation. Deletions between uORFs in the RSV leader decrease RSV RNA encapsidation to different extents RNA packaging in avian retroviruses requires specific cis- sequences located within the 5' leader (8-10). As the different Leader _s«L Genomic RNA mutants designed in this study are located near the proposed packaging signal (Fig. 3), we were interested to study the effect Probe of each deletion on the RSV RNA packaging. To this end, the Protected fragment mutations were inserted into the RSV genome (pAPr-C strain) and studied in vivo by transfecting the plasmids into chicken embryo fibroblasts (see Materials and Methods and ref. 18). The Figure 5. Viral RNA content of the virions produced in a transient transfection assay. At the top is represented a typicaJ RNase protection assay. Protected particles were purified and quantitated by. Western immuno- fragments have been analyzed by electrophoresis in a polyacrylamide blotting (Fig. 4). The RNA was extracted from an equivalent sequencing gel. Bands were visualized by autoradiography. The RNA for each amount of virions (normalized against the CA protein: Fig. 4) for virus (wild-type and mutants) was extracted from an equivalent number of each mutant and analyzed by RNase protection. A typical result virions (normalized to CA protein, Fig. 4). Names of the different virus are obtained with our packaging assays is shown in Figure 5. The indicated above the lanes. Lane WT/10 and WT/100 indicate 1:10 and 1:100 dilutions of RNA extracted from the WT virions, respectively. Lane C indicates mutant Del58, lacking a large portion of the U5 sequence, control cells. Undigested probes and protected fragments are indicated on the demonstrates a slight reduction in RNA packaging (40-50% as right. A schematic illustration of the probe used in this experiment is shown compared to wild-type). The Sel3 mutant carrying a lengthened below. The protected fragments covers the region from nucleoude positions version of the uORF3 ressembles the pAM3 22 4 mutant (described 630-256. The full-length probe contains an additional 75 nt from Bluescnpt plasmid (Stratagene). in ref. 18) and showed no defect in viral RNA packaging. Both mutants have an uORF3 which terminates in, or overlaps with the \\i packaging sequence without affecting RNA encapsidation. The mutant del 13 contains normal amount of viral RNA within the only 13 nt had a similar effect on translational activity at virion, while the Del44 and InsDel mutants packaged -5-10 % of AUGgag, as did deletion of 44 nt (a 2-fold increase in the viral RNA as compared as WT. This deletion encompasses production of luciferase compared to wt). nucleotides 160-167 which are part of a stem—loop structure that The presence of a new chimeric uORFl terminating at -7 0 nt has been recently shown to be crucial for viral RNA packaging upstream of AUG3 (Del58) increased translation at the AUGgag (38). Finally, removal of AUG3 plus the creation of a new AUG by 2-fold relative to wild-type (Fig. 3), as observed for Del44. The (M3189; Fig. 5) decrease packaging to >5% as already observed mutant M3 189 , with a new uORF3 situated 11 nt upstream of the for the pAM3 mutant lacking the third AUG (18). These results bona fide AUG3, also showed a 2-fold increase in translation. support the idea that sequences other than those already described Accordingly, this novel AUG3, although demonstrating a accept- are involved during the packaging step of the RSV infectious able sequence context, seems to be bypassed by ribosomes. These cycle. data emphasize the importance of the intercistronic interval between uORFl and 3 since a deletion as short as 13 nt impairs the ability of uORF3 to inhibit scanning ribosomes. The DISCUSSION intercistronic minimal distance which can be deleted could be shortened to 9 nt in the case of M3 189 , but because the AUG3 of In our previous study, we showed that mutations in the AUG this mutant is different than the WT AUG3, we cannot compare codons of the upstream open reading frames of RSV influence its intercistronic distance requirement to the other deletion translation and have a profound effect on the viral RNA mutants. packaging. Elucidation of the mechanism by which these uORFs influenced these processes required a better understanding of If the intercistronic distance were indeed the sole factor events occuring at each of the AUGs on the RSV leader. The influencing reinitiation at AUG3 or translational regulation at present study was aimed at defining more precisely the transla- g, then restoration of the wt intercistronic length should Downloaded from https://academic.oup.com/nar/article-abstract/23/5/861/2376433 by DeepDyve user on 06 August 2020 Nucleic Acids Research, 1995, Vol. 23, No. 5 867 tional events at the short open reading frames, using luciferase to be a sufficient interval, while in yeast, especially under starvation conditions and in plant cells, a longer spacing is fusion constructs. In addition, we designed specific deletions in preferred (26,40). In RSV, the distance requirement is between order to investigate the potential importance of reinitiation at that for yeast and preproinsulin and can be modulated depending AUG3 for translation. on the uORF present on the leader (see below). Here, we report that AUG1, which is the main ribosome binding site within the RSV leader (12,13), is also the most efficient Taken together with results from previous studies on RSV, our data are consistent with a role for uORFs in determining the translation initiation site (Fig. 1). This AUG, surrounded by a poor degree of reinitiation. Variations in the optimal intercistronic initiation context according to Kozak (31) is situated at an ideal distance can be seen in RSV. For example, the RSV mRNA consensus distance from the 5 ' cap (40 nt) and followed by a stable encoding the src gene, contains a 63 nt intercistronic distance secondary structure (36,37). These two factors are known to which is sufficient for alleviating the inhibitory effect of an uORF potentiate recognition at an AUG by scanning ribosomes (1). In (41). Thus, in die same virus and in the same cell (avian our fusion construct, we probably disrupted part of the secondary ftbroblasts), ribosomal subunits require different intercistronic structure which is possibly involved in assisting ribosomes to intervals to become competent for reinitiation (134 versus 63 nt). initiate at AUG 1. However, we found a 4-fold increase in initiation Differences in reinitiation competence have been well docum- at AUG 1 under these minimal conditions. It is therefore possible ented in me GCN4 gene of die yeast Saccharvmyces cerevisiae that measuring the heptapeptide in its natural context would show where it has been shown that certain intrinsic properties of an even greater stimulation. For uORF3, our data show a high uORF may be important in determining die efficiency of incidence of initiation at that AUG in the fusion study as well as downstream reinitiation and translation. The proximal region in the SeB mutant (and in ref. 18, pAM3 and pAMUP). The role close to the termination codon is crucial, in particular, A + U rich of AUG2 as an initiation site has been dismissed by our studies, but sequences in this region favor reinitiation (42—44). Nevertheless, this conclusion seems to disagree with the data from another lab it seems that this sequence determinant cannot be generalized to (29). It is worth noting that different avian strains are used (SR-A higher eukaryotic systems, since in RSV, the analogous region and Pr-C), although the sequence around the AUG2 is conserved surrounding die uORF stop codon is not A + U rich. The exact between both strains. However in their translational study which features of the uORFs that allow for this type of regulation remain showed the effect of uORF2 when it was elongated, Hackett and to be elucidated. collaborators used a truncated version of the RSV leader which In a recent report, we mentioned diat uORFs 1 and 3 regulate could be the source of the discrepancy between their results and RNA packaging, presumably through their translational prop- ours (29). Moreover, in an avian mutant (TK15), uORF2 is erties (18). The data presented here are in agreement widi such a elongated, overlapping the AUGgag. Translation was not affected role since both uORFl and 3 but not uORF2 are efficiently in this mutant, supporting the observation that AUG2 is not translated. With respect to the mechanism by which diese uORFs recognized by the ribosomes (39). act, one possibility is that ribosome pausing at AUG3 would So we can depict the following scheme for translation on RSV impede temporarily the flow of ribosomes on the RSV leader, leader: following translation of the first uORF, ribosomes mostly clearing the \y packaging sequence present in die leader just resume scanning to reinitiate at the third AUG situated 134 nt downstream of uORF3 (18); but this model is weakened by the further downstream (see Fig. 1: BsAUG31uc, BsORF31uc and leaky scanning occuring at AUG3 shown in this study (Fig. 3: Fig. 3, SeB). From the Sel3 mutant we can conclude that for two Sel3) Anodier possible mechanism that has been postulated ribosomes that initiate at AUG3, one passes through the uORF. recently involves the ability of the \\t sequence to form a Therefore both leaky scanning and reinitiation are involved in the stem-loop structure (7,38) which can be alternatively folded by translational regulation of RSV gene expression. Our results are base-pairing to uORF3 (7). The authors postulated that ribosomes consistent with AUG1 as the major translation initiation site on translating uORF3 sequence could negatively regulate packaging the RSV leader and AUG3 as being recognized with high by disrupting die secondary structure, but diey assumed that efficiency. This is similar to the GCN4 leader, however, in the AUG3 is poorly translated (29). Our data show direcdy that GCN4 system most ribosomes which reinitiate at downstream uORF3 is efficiendy translated (Fig. 1: BsAUG3 and BsORF31uc, uORFs dissociate from the mRNA (26). Whereas in RSV, after and Fig. 3: SeB). For this reason, we postulate that translation of translation of uORF3, ribosomes can still resume scanning to uORF3 is a prerequisite for packaging by the disruption of an reinitiate at AUGgag (Fig. 1 BsWT). inhibitory secondary structure, thus allowing binding of the In Rous sarcoma virus RNA, the distance between uORFl and Pr76gag to die \j/ region. This model could explain the uORF3 (134 nt) is well conserved between the different conservation of uORFs in die RSV leader rich in secondary avian/leukosis retrovirus strains (7,35). The conservation of the structure and diat efficient translation initiation of tiiese upstream intercistronic distance is consistent with our reinitiation model. AUGs supports a function in melting structured RNA (7,18). Indeed, as observed with deletion mutants (13 and 44 nt deletions), the intercistronic interval in RSV leader must be kept constant for efficient reinitiation at AUG3 and hence for ACKNOWLEDGEMENTS translational regulation at AUGgag. This agrees with other studies on reinitiation, where it was reported that the inhibitory We thank Andrew Craig, Greg Cosentino, Sylvie Mader and effect of inserting a single uORF on translation of downstream Robert Frederickson for critical reading of die manuscript We are product decreases as the uORF is moved further upstream (27). grateful to Nicholas Roggli for drawings and photographs and to To explain this observation, it was suggested that ribosomes Daniele Rifat for syntiiesis of oligonucleotides. One of us (OD) scanning from the uORF's termination codon to a downstream gratefully acknowledges the financial support of the Sandoz start codon require a certain period of time to bind a new set of Stiftung, Basel, Switzerland. This work was supported by grant initiation factors (34). In a mammalian system, 79 nt was found 31.29983.90 from the Swiss National Science foundation. Downloaded from https://academic.oup.com/nar/article-abstract/23/5/861/2376433 by DeepDyve user on 06 August 2020 868 Nucleic Acids Research, 1995, Vol. 23, No. 5 20 Meric, C. and SpahrP.F. (1986) J. ViroL, 60, 450-459. REFERENCES 21 De Wet, J.R., Wood, K.W., DeLuca, M., Helinski, D.R. and Subramani, S. (1987) Mol. Cell. BioL, 7, 725-737. 1 Kozak, M. (1989) J. Cell. BioL, 108, 229-241. 22 Oertle, S. and Spahr, P.F. (1990) J. Virol., 64, 5757-5763. 2 Merrick, W.C. (1992) Microbiol Rev., 56, 291-315. 23 Lo, K. M., Jones, S.S., Hackett, N. and Khorana, H.G. (1984) Proc. Natl. 3 Damiani, R.D. and Wessler, S.R. (1993) Prvc. Natl. Acad. ScL USA, 90, Acad. Sci. USA., 81, 2285-2289. 8244-8248. 24 Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492. 4 Degnin, C.R., Schleiss, M.R., Cao J. and Geballe, AP. (1993) J. ViroL. 67, 25 Hinnebusch, A.G. (1988) Proc. Natl. Acad Sci. USA, 85, 7279-7283. 5514-5521. 26 Abastado, J.P., Miller, P.F., Jackson, B.M. and Hinnebusch A.G. (1991) 5 Hill, J.R and Morris, D.R. (1993) J. BioL Chem., 268, 726-731. Mol. Cell. BioL, 11, 486-496. 6 Werner, M., Feller, A., Messenguy, F. and Pierard, A. (1987) Cell, 49, 27 Futterer, J. and Hohn, T. (1992) Nucleic Acids Res., 20, 3851-3857. 805-813. 28 Peabody, D.S. and Berg, P. (1986) Mot. Cell. BioL, 6, 2695-2703. 7 Hackett, P.B., Dalton, M.W., Johnson, D.P. and Petersen, R. (1992) Nucleic 29 Moustakas, A., Sonstegard, T.S. and Hackett, P.B. (1993) J. ViroL, 67, Acids Res., 19, 6929-6934. 4350-4357. 8 Linial, M. and MillerAD. (1990) Curr Topics Microbiol. Immunol., 157, 30 Kozak. M. (1986) Proc. Natl. Acad. Sci. USA, 83, 2850-2854. 125-152. 31 Kozak, M. (1991) J. Cell. BioL, 115, 887-903. 9 Kate, R.A., Terry, R.W. and Skalka, A.M. (1986) J. ViroL, 59, 163-167. 32 Pelletier, J. and Sonenberg, N. (1985) Cell, 40, 515-526. 10 Aronoff, R., Hajjar, AM. and Linial, M.L. (1993) J. Virol., 67, 178-188. 33 Pelletier, J. and Sonenberg, N. (1988) Nature, 334, 320-325. 11 Darlix, J.L., Spahr, PR, Bromley, P.A. and Jaton, J.C. (1979) / . Virol., 29, 34 Kozak, M. (1987) Mol. Cell. BioL, 7, 3438-3445. 597-611. 35 Bizub, D., Katz, R. A. and Skalka, A.M. (1984) / Virol, 49, 557-565. 12 Petersen, R. B., Hensel, CM . and Hackett, P.B. (1984) J. Virol., 51, 36 Cobrinik, D., Sosky, L. and Leis, J. (1988) J. ViroL, 62, 3622-3630. 727-729. 37 Cobrinik, D., Aiyar, A., Ge, Z., Katzman, M., Huang, H. and Leis, J. 13 Petersen, R. B. and Hackett, P.B. (1985) J. Virol, 56, 683-690. (1991) J. ViroL, 65, 3864-3872. 14 Hackett, P. B., Petersen, R. B. , Hensel, C. H. , Albericio, F., Gunderson, S. 38 Knight, J.B., Si, Z.H and Stoltefus, CM . (1994) J. Virol, 68, 4493-4502. I., Palmenberg, A. C. and Barany, G. (1986) J. Mol. BioL, 190, 45-57. 39 Kawai, S and T. Koyama. (1984) J. ViroL, 51, 147-153. 15 Hensel, C. H., Petersen, R. B. and Hackett, P.B. (1989) / . ViroL, 63, 40 Futterer, J. and Hohn, T. (1991) EMBO J., 10, 3887-3896. 4986-4990. 41 Hughes, S., Mellstrom, K., Kosik, E., Tamanoi, F. and Brugge, J. (1984) 16 Moustakas, A., Sonstegard, T.S. and Hackett, P.B. (1993) J. ViroL, 67, Mol. Cell BioL, 4, 1738-1746. 4337-4349. 42 Grant, CM . and Hinnebusch, A.G. (1994) Mol. Cell. BioL, 14, 606-618. 17 Petersen, R. B., Moustakas, A. and Hackett, P.B. (1989) J. ViroL, 63, 43 Miller, P. F. and Hinnebusch, A.G. (1989) Genes Dev., 3, 1217-1225. 4787^796 . 44 Mueller, P.F, Jackson, B.M., Miller, P.F. and Hinnebusch, A.G. (1988) 18 Donze", O. and Spahr, P.F. (1992) EMBO J., 11, 3747-3757. Mol. CelL BioL, 8, 5439-5447. 19 Sambrook, J., Fristch E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor University Press, Cold Spring Harbor, NY. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

The first and third uORFs in RSV leader RNA are efficiently translated: implications for translational regulation and viral RNA packaging

Download PDF
8 pages

Loading next page...
 
/lp/oxford-university-press/the-first-and-third-uorfs-in-rsv-leader-rna-are-efficiently-translated-ImnUVmx5Mo

References (0)

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Oxford University Press
Copyright
© 1995 Oxford University Press
ISSN
0305-1048
eISSN
1362-4962
DOI
10.1093/nar/23.5.861
Publisher site
See Article on Publisher Site

Abstract

Downloaded from https://academic.oup.com/nar/article-abstract/23/5/861/2376433 by DeepDyve user on 06 August 2020 11995 Oxford University Press Nucleic Acids Research, 1995, Vol. 23, No. 5 861-868 The first and third uORFs in RSV leader RNA are efficiently translated: implications for translational regulation and viral RNA packaging Olivier Donz6*, Pascal Damay and Pierre-Francois Spahr Department of Molecular Biology, University of Geneva, 30 Quai E. Ansermet, 1211 Geneva 4, Switzerland Received September 1,1994; Revised and Accepted January 24,1995 ABSTRACT These three elements are situated at different strategic places within the leader. The first and second uORFs are found within Rous sarcoma virus (RSV) RNA leader contains three the proximal 5' secondary structure-rich region while the third short upstream open reading frames. We have shown uORF is located close to the packaging signal (termed y) , which recently that both uORFs 1 and 3 Influence in vivo is required for efficient recognition of the viral RNA by translation of the downstream gag gene and are /ra/u-acting factors (8-10). Previous studies have shown that involved in the virus RNA packaging process. In this AUG1 is the main ribosome binding site in the RSV leader report, we have studied the translational events (11-13) and that the encoded heptapeptide product is synthesized occurlng at the upstream AUGs in vivo. We show that in vitro (14). Furthermore, mutations that alter the RSV leader (i) the first and third AUGs are efficient translational AUGs increased downstream translation in vitro only slightly initiation sites; (II) ribosomes reinitiate efficiently at (15), yet have profound effects on viral replication (16—18). In a AUG3; and (Hi) deletions in the intercistronlc distance recent study, we characterized the role of the three uORFs present between uORF1 and 3 (which is well conserved among in the leader of Rous sarcoma virus (Prague C strain). We reported avian strains) prevent ribosome Initiation at AUG3, that uORFl and 3 are key elements involved in the viral life cycle thus Increasing translation efficiency at the down- and act by regulating the efficiency of translation at the stream AUGgag. The roles of the uORFs in translation downstream AUGgag as well as the efficiency of viral RNA and packaging are discussed. packaging (18). Mutation of either AUG1 or 3 has a profound effect on RNA encapsidation, inhibiting this process by INTRODUCTION 20-50-fold. We proposed a model whereby ribosomes first translate uORFl and then subsequently reinitiate and pause at the In eukaryotic cells, translation is usually initiated in a manner AUG3. We postulated that initiation at the third AUG is the consistent with the ribosome scanning model (1). According to central step in the regulation of RNA packaging. Reinitiation at this model, the 40S ribosomal subunit and translation initiation AUG3 would impede temporarily the flow of ribosomes on the factors bind to the 5 ' end of mRNA and scan the RNA leader until RSV leader, clearing the \\f packaging sequence present in the they reach an AUG codon in the appropriate context. Subsequent- leader just downstream of uORF3 (18). ly, the 60S ribosome joins and initiates polypeptide chain In the present study, we examined this prediction of our elongation (2). The scanning model accounts for the effects of proposed model in vivo. To this end, we looked at the translation structural features within the 5' untranslated region, such as initiation rates at each AUG present within the RSV leader RNA. secondary structure and open reading frames, which can strongly The reporter gene firefly luciferase was fused to all constructs to influence the efficiency of translation initiation. The presence of allow quantitation of translational initiation rates at the different one or more upstream open reading frames (uORFs) has been AUGs. The reinitiation hypothesis between uORFl and 3 was shown to influence the translation of downstream ORFs. also tested by altering the intercistronic distance. Our data reveal Initiation occurs preferentially at the upstream site, which in turn that uORFl and 3 are efficient initiation sites for translation and reduces initiation downstream due to the apparent inefficiency of strongly suggest that AUG3 is recognized by reinitiating reinitiation at internal AUG codons. Therefore, in most cases, ribosomes. We also showed the effects of these RSV RNA leader uORFs inhibit downstream translation in proportion to the alterations on the efficiency of viral RNA packaging. efficiency of their own translation (1). In a few cases, the coding capacity of the uORF was found to influence the downstream translation (3-6). MATERIALS AND METHODS In Rous sarcoma virus, the 5 ' leader contains three short open Cell culture reading frames of 7, 16 and 9 codons in length. The three uORFs are conserved in length, and in the nucleotide sequence surround- Chicken embryo fibroblasts prepared from Spafas eggs (Gs~ and ing the initiation codons among the avian/leukosis viruses (7). Chf~: Norwich, CT, USA) were grown in Dulbecco-modified *To whom correspondence should be addressed at present address: Department of Biochemistry, McGill University, 3655 Drummond Street, Montreal, Quebec H3G 1Y6, Canada Downloaded from https://academic.oup.com/nar/article-abstract/23/5/861/2376433 by DeepDyve user on 06 August 2020 862 Nucleic Acids Research, 1995, Vol. 23, No. 5 Eagle medium containing 5% fetal calf serum (Gibco Labora- performed the same scheme to construct InsDel from BsDel44. tories, Grand Island, NY) at 41 °C in an atmosphere supplem- The same pSp73 insert was used and again a double insert was ented with 5% CO2. present in the construct, but in the same orientation. InsDel carries, thus, a 94 nt insert at position 102 in addition of the 44 nt deletion: the intercistronic distance in this construct is 184 nt Bacterial strains instead of 134 nt. In Del58, we removed the 58 nt RsrU-BstEll Escherichia coli DH5cc and CJ236 were grown according to the fragment (positions 47 and 102, respectively). Del 13 was instructions of the mutagenesis kit (Biorad). E.coli DH5a was constructed by digesting the Bslead clone with BstEll followed rendered competent as previously described (19). Plasmid DNAs by treatment with mung Bean nuclease. We obtained a 13 nt were purified from either small or large bacterial cultures by the deletion from nucleotides 96 to 108. The introduction of the alkaline lysis method and for transfection were further purified by mutations was confirmed by the dideoxy-chain termination precipitation with polyethylene glycol (PEG) (19). method of DNA sequencing using T7 polymerase (Pharmacia) primed by a synthetic oligonucleotide complementary to the 3' Cloned DNAs end of the RSV leader. The mutated fragments were cloned back into pAsPrc using the Sphl sites and then the SaH-EcoRN Plasmid pAPrc has already been described (20); it contains a fragment was introduced into pAPrc (20). non-permutated copy of the provirus RSV Prague C strain. Plasmid pAsPrc is a Sall-EcoRV subclone of pAPrc in pBR322 Transfection containing the entire leader and gag sequences. All the mutations were constructed in Bslead, a 1167 bp Sphl fragment of pAsPrc Cells either freshly prepared from embryos or frozen in the cloned in the phagemid vector Bluescribe (-) (Stratagene, San presence of glycerol were used for transfection after two to seven Diego, USA). passages. Cells were transfected using the DEAE-Dextran To construct the plasmid carrying the luciferase gene (Bsluc), procedure as described previously (18). the Bsml-BamUl fragment (with the ends filled by the Klenow fragment of DNA polymerase I) from pRSVluc (21) containing Protein analysis the luciferase gene was cloned by blunt end ligation into Viral particles produced by the transfected cells were purified by BsleadMin to replace the HindUl fragment encoding the gag gene ultracentrifugation through a 20% sucrose cushion and their (the HindJQ ends were similarly filled by Klenow prior to protein content analyzed by immunoblotting with polyclonal ligation). BsleadMin carries the 1167 bp Sphl fragment of pAsPrc antibodies against RSV CA (p27), as described previously (20). in which the AUG initiator of the gag gene at position 380 has been mutated to TTC in order to create a HindUl site at the Luciferase assay beginning of the gag gene (22). Bsluc contains the RSV leader fused to the luciferase gene, including 20 nucleotides (nt) of the Each 10 cm plate of transfected CEF was washed three times in luciferase leader. A 440 nt Pstl fragment from each Bslead mutant phosphate-buffered saline without Ca2+ and the cells were was cloned into the corresponding site of Bsluc to replace the harvested in 500 (il of lysis buffer (Promega). Cell debris was sequence derived from BsleadMin. pelleted by centrifugation in a microcentrifuge for 5 min at 4°C. A 20 \i\ aliquot of extract was added to 100 u.1 of assay buffer Construction of mutants in RSV leader (Promega) in a small test tube. Luciferase activity was measured in millivolts in a luminometer (Bioorbit) using the luciferase The following oligonucleotides were synthesised on an Applied assay system (Promega). Biosystems 381 A DNA synthetizer and purified as previously Total luciferase RNA was isolated from transfected CEF using described (23): the guanidinium/CsCl method (19). RNA was then digested with DNase I and subjected to a RNase ONE protection (Promega) M3189: 3'-GCAGAGCGAATAAGCrCCTACCCrGCAGTTGGGAT- because RNA levels were too low for decisive Northern CATCTCCCCC-5' experiments. Prior to conducting the experiments shown.in SEL3:3'-CTCCCCCGACGCCGAAATCCTCCCGTCTTC-5' Figures 2 and 5, we evaluated this 'RNase One' by using different Del44:3'-ATCAATCCCTTATCACCAAAGCCCCTCGCC-5'par amounts of pure luciferase RNA (from Promega). The data from ORFl-luc: 3'-AACTACCGGCCTGGCAGCTAAGGGCTTCTGCG- this experiment convinced us that this enzyme gave a linear and GTTTTTGTAT-5' quantitative signal, although residual undigested probe was still ORF2-luc: 3'-CTGGGGCTGCACTATCMTCCCTTCTGCGGTnT- present. To obtain protected fragment of the same size for TGTATTTC-5' luciferase constructs, a 413 RNA antisense probe was synthesized ORF3-luc: 3'-TGGGATCATCTCCCCCGACGCCGACrTCTGCGG- from the pGEM-luc (Promega) digested with £coRV; this probe TTTTTGTATTTC-5' contains homologies to the last 356 nt of the luciferase ORF. AUG31uc: 3'-GCGAATAAGCCCCTCGCCTGCTACCTTCTGCGG- TTTTTGTATTTCTTTCCG-5' Purification of viral RNA AH the mutants cited above were constructed as described previously (24). The viral RNA of virions produced after transfection was purified The mutant Ins was made by digesting the Bslead clone (20) and analyzed by RNase protection assay. The RNA was extracted from virions as described previously( 18). Total cellular RNA was with BstEU (position 102). A BglU-SaCl insert (from plasmid purified from subconfluent cultures by lysis in guanidinium pSp73: Promega) was treated with mung bean nuclease and ligated to Bslead to give pAsIns: two inserts in inverted thiocyanate, followed by centrifugation through cesium chloride orientation are present, adding 94 nt to the RSV leader. We as described (19). Downloaded from https://academic.oup.com/nar/article-abstract/23/5/861/2376433 by DeepDyve user on 06 August 2020 Nucleic Acids Research, 1995, Vol. 23, No. 5 863 own Bs WT luciferase Bs ORF1 luciferase . Bs ORF2 tuciferase , 0RF2 0RF3 tucfftrss* Bs ORF3 luciferase , 7777X AUO 0RF1 0RF2 AU63 \ixH&r%%m Bs AUG3 luciferaie, 1 i i Relative Ljdferase activity Figure 1. Efficiency of translation initiation at the different AUGs present in RSV leader. The schematic depicts the 5' end encoded by the different luciferase fusion construct (drawn to scale). The uORFs are represented by boxes. The hatched boxes symbolize the uORFs involved in translation and packaging rcgulation (9). The numbers indicate the first AUG triplet for each uORF-luciferase fusion and the AUG initiator of the luciferase gene. Levels of luciferase activity are shown at the right and arc given relative to the control plasmid with the whole RSV leader (BsWT-luciferase); values have been normalized to tbe RNA levels (Fig. 2). The light units produced by any given luciferase expression vector varied by <10% in parallel transfections using two independent clones for each construct The emitted light was quantitated in a Bioorbit luminometer. Quantitation of RNA present in the virions or in the cell were luciferase activities observed were due to differential transla- performed using an RNase-protection assay. Plasmid pL(-), tional efficiencies rather than transcriptional variations, we which contains the EcoRl-Xhol fragment from pAPrC (20), was monitored levels of luciferase RNA within the transfected cells by digested with Sad and in vitro transcribed using T7 polymerase RNase protection analysis. A representative protection experi- and a commercial kit (Promega) according to the kit instructions. ment is shown in Figure 2, using a probe which protected 356 nt The antisense RNA probe contains homologies to 355 nt of the of the luciferase mRNA. In several experiments, we observed leader and the gag gene. Plasmid pL(-) was also digested with similar levels of RNA synthesized from each construct, indicating BstEU and in vitro transcribed using T7 polymerase. The that the variations in luciferase activity truly reflected altered antisense transcript produced was used to detect the presence of translational efficiencies of the recombinant RNAs. the mutations in viral RNA extracted from the different mutant The luciferase activities in extracts from cells transfected with virions (data not shown). RNase protection was performed with each construct were normalized to the activity in cells transfected RNase One (Promega) as described by the manufacturer. The with BsWT-luciferase construct, which contained the whole RSV nuclease resistant hybrid was analysed on a denaturing polyacryl- leader fused to the luciferase sequence (Fig. 1 and ref. 18). amide gel and the product detected by autoradiography. Translational efficiency at the first AUG was measured in the BsORFl-luciferase plasmid which produced as much as 4-fold RESULTS more activity than at the AUGgag (compare Bswtluc with BsORFlluc in Fig. 1). The luciferase activity present in the lysate Upstream AUG codons 1 and 3 in RSV leader RNA are of cells transfected with BsORF2-luciferase was no more than 3% efficient initiation sites of the activity observed at the AUGgag and even less if we compare to the efficiency at AUG1. This indicates that AUG2 is We showed previously that alterations of AUG 1 or AUG3 present poorly recognized by ribosomes during the scanning process, in in the RSV leader RNA influences translation of the gag gene, agreement with previous observations (18,29). To investigate situated downstream of the uORFs. uORFl is a translational initiation occuring at AUG3, we used two different fusion enhancer of viral proteins, while uORF3 inhibits downstream plasmids (Fig. 1): in the BsORF3-luciferase construct, the translation at the AUGgag (18). These results strongly suggested luciferase was fused to the end of the third reading frame, whereas that ribosomes initiate and translate both uORFl and uORF3 in in the BsAUG3-luciferase plasmid, the reporter sequence was vivo. To study further the translational events occuring at the RSV directly fused to AUG3. Both constructs showed that AUG3 is an leader uORFs, we fused the end of each uORF to the coding efficient initiation codon (2-fold increase compared to AUGgag sequence of the firefly luciferase gene (21). Fusion constructs in Bswtluc; Fig. 1). Although a precise comparison of the levels have been successfully used for a better understanding of the uORFs present in the leader of the GCN4 gene in the yeast of expression of the fusion proteins could not be made without Saccharomyces cerevisiae (25). Since the luciferase coding knowledge of differences in the stability and specific activity of region was fused at the end of the uORFs in order to minimize each, the results support the idea that both uORFl and uORF3 are artifacts due to the fused gene, we reasoned that expression of the sites for efficient translation initiation. uORF-luciferase fusions should reflect the translational rates of To rule out the possibility that luciferase activities observed unmodified uORFs (see Discussion). For each construct, we used were due to recognition of a cryptic initiation codon present two independent clones. The expression from each fusion within the luciferase coding region (21) rather than those within construct was tested by transient transfection into chicken the RSV leader region, we mutated the initiation AUG into a embryo fibroblasts (CEFs). To ensure that differences in non-initiation codon for each uORF fusion construct. The control Downloaded from https://academic.oup.com/nar/article-abstract/23/5/861/2376433 by DeepDyve user on 06 August 2020 864 Nucleic Acids Research, 1995, Vol. 23, No. 5 AUG codon (26-28). This was ascertained by studying the luciferase gene fused to the RSV leader. The Sel3 mutation did not affect the mRNA level as observed by RNase protection analysis (Fig. 2). As expected, the lengthened version of uORF3 affected the translational efficiency at AVGgag, causing a 3-fold decrease in luciferase activity compared to wild-type value (35% -* - Probe translation efficiency compared to WT) (Fig. 3). Together with -* - Protected results of uORF-luciferase fusion studies, these data point out the fragment strong initiation potential of the third AUG, contradicting a previous study which showed indirectly that AUG3 is initiated to a small extent (29). Insertion of a sequence with the potential to form stable secondary structures greatly inhibits protein expression initiated at AVGgag The presence of a sequence with the potential to form stable secondary structures in the 5' mRNA leader generally inhibits translation in eukaryotes, presumably because it interferes with the scanning process (30-32). In the case of poliovirus mRNA, — - Probe evidence was presented that these negative elements are without —•- Protected effect as the mRNA is initiated by the process of internal initiation fragment (33). To determine whether ribosomes can overcome such an inhibitory element in the RSV leader, we introduced a sequence capable of forming a stable secondary structure between uORFl and uORF3 (mutant 'Ins' Fig. 3). This sequence was extremely stable (>150 Kcal/mol) and was formed by a 94 nt insert with dyad symmetry. When transcribed into mRNA, it is predicted to form a stable hairpin structure. We introduced this sequence at S'c*p position 102 within the RSV leader which is 38 nt downstream EcoH Ludferai e mRNA IUO from the uORFl termination codon and 96 nt upstream from the 4l3nt -* - Probe uORFi initiation site. Sequences with similar predicted second- ary structure have been shown to reduce greatly cap-dependent Protected fragment translation in eukaryotic cells (30,32). As shown in Figure 3 (compare WT with Ins), the presence of this stable structure within the leader strongly inhibits translation initiation at the Figure 2. RNase protection analysis of luciferase containing mRNAs produced by the different Bsluciferase constructs. On the top is the analysis of protected AVGgag (>1% of WT). The observed defect was manifested at fragments by electrophorcsis in a polyacrylamide sequencing gel. Bands were the level of translation, since analysis of mRNA showed similar visualized by autoradiography. Names of the different constructs designed in levels of transcript (Fig. 2). To test whether this insertion was Figures I and 3 are shown above the corresponding lane. Undigested probes inhibitory due to the stem structure or simply because of its and protected fragments are indicated on the right. At the bottom is shown a schematic illustration of the probe used in this experiment The £coRV particular sequence, we inserted the same sequence without dyad represents the 3' end of the RNA probe. symetry in the same location (see Materials and Methods and Fig. 3: InsDel). The presence of this unstructured sequence does not affect translational efficiency at the AVGgag. This indicates that constructs were transfected into CEFs and luciferase activity was secondary structure in the Ins construct was responsible for.the monitored in the lysate of transfected cells. None of the cells observed effect, rather than the sequence itself (compare Ins and transfected with control plasmids produced any detectable InsDel: Figs 3 and 4). These data, taken together with the luminescer.ee and as such were comparable to the luciferase observations reported above, strongly suggest that translation of activity from lysates of the mock-transfected cells (data not uORFs and of the gag gene occurs in vivo by a ribosome scanning shown). This demonstrated that the luciferase data reported above along the RSV leader. These data also fit with previous results reflect the use of the RSV leader AUGs. showing that in vitro a stable secondary structure present within the RSV leader decrease translation to 10% compared to the WT Elongation of uORF3 diminished translation at the level (15). AVGgag To investigate whether the initiation potential of AUG3 in the Effect of intercistronic length between uORFl and 3 on full-length virus leader parallels that of the luciferase-fusion downstream translation construct, we lengthened uORF3 in the RSV leader by addition of a single base (a thymidine) at the end of uORF3 (Sel3: Fig. 3). Efficient reinitiation is dependent on intercistronic length. It has This addition shifts the frame of the third uORF, leading to been observed that inhibition of preproinsulin synthesis by an termination 11 codons downstream of the AVGgag. Since uORF increased as the intercistronic distance was decreased from uORF3 in Sel3 overlaps the gag sequence, translation initiation 79 to 2 nt (34). In our system, the uORFl and 3 are separated by at AUG3 codon is expected to interfere with initiation at the gag 134 nt and this interval is well conserved between the different Downloaded from https://academic.oup.com/nar/article-abstract/23/5/861/2376433 by DeepDyve user on 06 August 2020 Nucleic Acids Research, 1995, Vol. 23, No. 5 865 WT Sel 3 ORFJ V//A M31" Aue Vc* OBF3 AUSn aian *U0« Ael58 0BF3 AUS H V///A Ael13 ORF3 V///A InsAel 0KF3 lom Y///A Ins 0 1 2 3 Relative Uidferase activity Figure 3. Translational efficiency of the mutants in the RSV leader. The scheme shows the different mutants with alterations in the intercistronic distance between uORFl and uORF3 as well as in uORFl and uORF3. The functional uORFs are represented in hatched boxes. Levels of luciferase activity are given relative to the control plasmid with the whole RSV leader (BsWT-luciferase). The light units produced by any given luciferase expression vector varied by <1O% in parallel transfections (see Materials and Methods). the surrounding sequences are dispensable for regulation of both translation and packaging (36,37). Restriction sites present within the first (RsrQ) and second uORFs (BstEU) were used to delete 58 nt and to create a new uORF beginning at AUG1 and terminating at the uORF2 stop codon. This mutant (Del58) possesses an uORFl of 12 codons terminating at position 130, thus situated at 68 nt upstream of the third initiation codon. For mutant M3' 89 , AUG 3 was mutated to UCA (as in pAM3:18) and CA— a new AUG (in a good context for translation: AGGATGG) was created at position 189 to replace an AGC codon. In the latter case, translation of the new uORF3 would occur in a - 1 frame producing a pentapeptide product. Moreover, the intercistronic interval was decreased by 11 nt in this mutant The mutants Del44 and Del 13 did carry intact uORFl and 3, while the two other mutants, Del58 and M3189 have a modification in uORFl or Figure 4 . Analysis of the virion gag-encoded proteins. Virions produced by the uORF3, respectively. transfected cells were purified as described in Materials and Methods. Viral To evaluate the effect of these deletions on translation initiation proteins were resolved by SDS-PAGE and immunoblotted with polyclonal at the AUGgag codon in vivo, we measured the emission of light antibodies against RSV CA (p27) and detected with 123I-labelled protein A. produced by the luciferase fusion constructs under the control of The mutants designated in Figure 3 are indicated above each lane. Lane C indicates the control cells, mock transfected with no DNA. the AUGgag codon (Fig. 3). For each construct, two independent clones were used, giving identical results. Moreover, in several experiments, we showed that mRNAs of the mutants were avian strains (7,35). If reinitiation occurs in the RSV leader, then transcribed to similar levels (Fig. 2). decreasing the distance between uORFl and uORF3 should allow The 44 nt deletion (Del44) led to a 2-fold increase in ribosomes to bypass the uORF3, thus increasing translation translational efficiency at the downstream AUG gag codon. The efficiency downstream at the AUGgag, as observed in the enhanced translation produced by this deletion mimics the effect absence of AUG3 (18). To test this possibility, several mutants caused by mutation of AUG3 (2-fold increase; 18). One possible with decreasing distance between both uORFs were made and interpretation of this result is that the intercistronic interval is too two independent clones were used for each mutant (Fig. 3). The short to allow efficient reinitiation at AUG3 (34), thus permitting first construct (Del44) has a 44 nt deletion between the the ribosomes to initiate downstream at the AUGgag more minicistrons (positions 137-181). Another carries a 13 nt deletion efficiently. More surprising was the result obtained for Del 13: within the uORF2 (Del 13); we chose to delete this region because shortening of the intercistronic space between uORFl and 3 by Downloaded from https://academic.oup.com/nar/article-abstract/23/5/861/2376433 by DeepDyve user on 06 August 2020 866 Nucleic Acids Research, 1995, Vol. 23, No. 5 allow ribosomes to initiate at AUG3, thereby inhibiting transla- tion initiation at AUGgag. To test this prediction, we inserted 94 nt into Del44 between uORFl and uORF3 at a place devoid of assigned function (position 102; see InsDel: Fig. 3). Thus, this leader carries a 94 nt insertion coupled with a 44 nt deletion, to rebuild an intercistronic distance greater than the wt distance. The new hybrid intercistronic interval restored completely the rein- • *— •+— Probe itiation competence of the ribosomes to wild-type level (Fig. 3), revealing that a longer distance between uORFl and uORF3 does - Protected not favor more efficient reinitiation. Accordingly, 134 nt appears fragment to be the preferred reinitiation distance for the virus: increasing it does not enhance reinitiation efficiency, whereas deletion of only 13 nt strongly impairs reinitiation. The experiments presented here are consistent with the idea that reinitiation is distance-dependent and strongly suggest that reinitiation is involved in RSV translational regulation. Deletions between uORFs in the RSV leader decrease RSV RNA encapsidation to different extents RNA packaging in avian retroviruses requires specific cis- sequences located within the 5' leader (8-10). As the different Leader _s«L Genomic RNA mutants designed in this study are located near the proposed packaging signal (Fig. 3), we were interested to study the effect Probe of each deletion on the RSV RNA packaging. To this end, the Protected fragment mutations were inserted into the RSV genome (pAPr-C strain) and studied in vivo by transfecting the plasmids into chicken embryo fibroblasts (see Materials and Methods and ref. 18). The Figure 5. Viral RNA content of the virions produced in a transient transfection assay. At the top is represented a typicaJ RNase protection assay. Protected particles were purified and quantitated by. Western immuno- fragments have been analyzed by electrophoresis in a polyacrylamide blotting (Fig. 4). The RNA was extracted from an equivalent sequencing gel. Bands were visualized by autoradiography. The RNA for each amount of virions (normalized against the CA protein: Fig. 4) for virus (wild-type and mutants) was extracted from an equivalent number of each mutant and analyzed by RNase protection. A typical result virions (normalized to CA protein, Fig. 4). Names of the different virus are obtained with our packaging assays is shown in Figure 5. The indicated above the lanes. Lane WT/10 and WT/100 indicate 1:10 and 1:100 dilutions of RNA extracted from the WT virions, respectively. Lane C indicates mutant Del58, lacking a large portion of the U5 sequence, control cells. Undigested probes and protected fragments are indicated on the demonstrates a slight reduction in RNA packaging (40-50% as right. A schematic illustration of the probe used in this experiment is shown compared to wild-type). The Sel3 mutant carrying a lengthened below. The protected fragments covers the region from nucleoude positions version of the uORF3 ressembles the pAM3 22 4 mutant (described 630-256. The full-length probe contains an additional 75 nt from Bluescnpt plasmid (Stratagene). in ref. 18) and showed no defect in viral RNA packaging. Both mutants have an uORF3 which terminates in, or overlaps with the \\i packaging sequence without affecting RNA encapsidation. The mutant del 13 contains normal amount of viral RNA within the only 13 nt had a similar effect on translational activity at virion, while the Del44 and InsDel mutants packaged -5-10 % of AUGgag, as did deletion of 44 nt (a 2-fold increase in the viral RNA as compared as WT. This deletion encompasses production of luciferase compared to wt). nucleotides 160-167 which are part of a stem—loop structure that The presence of a new chimeric uORFl terminating at -7 0 nt has been recently shown to be crucial for viral RNA packaging upstream of AUG3 (Del58) increased translation at the AUGgag (38). Finally, removal of AUG3 plus the creation of a new AUG by 2-fold relative to wild-type (Fig. 3), as observed for Del44. The (M3189; Fig. 5) decrease packaging to >5% as already observed mutant M3 189 , with a new uORF3 situated 11 nt upstream of the for the pAM3 mutant lacking the third AUG (18). These results bona fide AUG3, also showed a 2-fold increase in translation. support the idea that sequences other than those already described Accordingly, this novel AUG3, although demonstrating a accept- are involved during the packaging step of the RSV infectious able sequence context, seems to be bypassed by ribosomes. These cycle. data emphasize the importance of the intercistronic interval between uORFl and 3 since a deletion as short as 13 nt impairs the ability of uORF3 to inhibit scanning ribosomes. The DISCUSSION intercistronic minimal distance which can be deleted could be shortened to 9 nt in the case of M3 189 , but because the AUG3 of In our previous study, we showed that mutations in the AUG this mutant is different than the WT AUG3, we cannot compare codons of the upstream open reading frames of RSV influence its intercistronic distance requirement to the other deletion translation and have a profound effect on the viral RNA mutants. packaging. Elucidation of the mechanism by which these uORFs influenced these processes required a better understanding of If the intercistronic distance were indeed the sole factor events occuring at each of the AUGs on the RSV leader. The influencing reinitiation at AUG3 or translational regulation at present study was aimed at defining more precisely the transla- g, then restoration of the wt intercistronic length should Downloaded from https://academic.oup.com/nar/article-abstract/23/5/861/2376433 by DeepDyve user on 06 August 2020 Nucleic Acids Research, 1995, Vol. 23, No. 5 867 tional events at the short open reading frames, using luciferase to be a sufficient interval, while in yeast, especially under starvation conditions and in plant cells, a longer spacing is fusion constructs. In addition, we designed specific deletions in preferred (26,40). In RSV, the distance requirement is between order to investigate the potential importance of reinitiation at that for yeast and preproinsulin and can be modulated depending AUG3 for translation. on the uORF present on the leader (see below). Here, we report that AUG1, which is the main ribosome binding site within the RSV leader (12,13), is also the most efficient Taken together with results from previous studies on RSV, our data are consistent with a role for uORFs in determining the translation initiation site (Fig. 1). This AUG, surrounded by a poor degree of reinitiation. Variations in the optimal intercistronic initiation context according to Kozak (31) is situated at an ideal distance can be seen in RSV. For example, the RSV mRNA consensus distance from the 5 ' cap (40 nt) and followed by a stable encoding the src gene, contains a 63 nt intercistronic distance secondary structure (36,37). These two factors are known to which is sufficient for alleviating the inhibitory effect of an uORF potentiate recognition at an AUG by scanning ribosomes (1). In (41). Thus, in die same virus and in the same cell (avian our fusion construct, we probably disrupted part of the secondary ftbroblasts), ribosomal subunits require different intercistronic structure which is possibly involved in assisting ribosomes to intervals to become competent for reinitiation (134 versus 63 nt). initiate at AUG 1. However, we found a 4-fold increase in initiation Differences in reinitiation competence have been well docum- at AUG 1 under these minimal conditions. It is therefore possible ented in me GCN4 gene of die yeast Saccharvmyces cerevisiae that measuring the heptapeptide in its natural context would show where it has been shown that certain intrinsic properties of an even greater stimulation. For uORF3, our data show a high uORF may be important in determining die efficiency of incidence of initiation at that AUG in the fusion study as well as downstream reinitiation and translation. The proximal region in the SeB mutant (and in ref. 18, pAM3 and pAMUP). The role close to the termination codon is crucial, in particular, A + U rich of AUG2 as an initiation site has been dismissed by our studies, but sequences in this region favor reinitiation (42—44). Nevertheless, this conclusion seems to disagree with the data from another lab it seems that this sequence determinant cannot be generalized to (29). It is worth noting that different avian strains are used (SR-A higher eukaryotic systems, since in RSV, the analogous region and Pr-C), although the sequence around the AUG2 is conserved surrounding die uORF stop codon is not A + U rich. The exact between both strains. However in their translational study which features of the uORFs that allow for this type of regulation remain showed the effect of uORF2 when it was elongated, Hackett and to be elucidated. collaborators used a truncated version of the RSV leader which In a recent report, we mentioned diat uORFs 1 and 3 regulate could be the source of the discrepancy between their results and RNA packaging, presumably through their translational prop- ours (29). Moreover, in an avian mutant (TK15), uORF2 is erties (18). The data presented here are in agreement widi such a elongated, overlapping the AUGgag. Translation was not affected role since both uORFl and 3 but not uORF2 are efficiently in this mutant, supporting the observation that AUG2 is not translated. With respect to the mechanism by which diese uORFs recognized by the ribosomes (39). act, one possibility is that ribosome pausing at AUG3 would So we can depict the following scheme for translation on RSV impede temporarily the flow of ribosomes on the RSV leader, leader: following translation of the first uORF, ribosomes mostly clearing the \y packaging sequence present in die leader just resume scanning to reinitiate at the third AUG situated 134 nt downstream of uORF3 (18); but this model is weakened by the further downstream (see Fig. 1: BsAUG31uc, BsORF31uc and leaky scanning occuring at AUG3 shown in this study (Fig. 3: Fig. 3, SeB). From the Sel3 mutant we can conclude that for two Sel3) Anodier possible mechanism that has been postulated ribosomes that initiate at AUG3, one passes through the uORF. recently involves the ability of the \\t sequence to form a Therefore both leaky scanning and reinitiation are involved in the stem-loop structure (7,38) which can be alternatively folded by translational regulation of RSV gene expression. Our results are base-pairing to uORF3 (7). The authors postulated that ribosomes consistent with AUG1 as the major translation initiation site on translating uORF3 sequence could negatively regulate packaging the RSV leader and AUG3 as being recognized with high by disrupting die secondary structure, but diey assumed that efficiency. This is similar to the GCN4 leader, however, in the AUG3 is poorly translated (29). Our data show direcdy that GCN4 system most ribosomes which reinitiate at downstream uORF3 is efficiendy translated (Fig. 1: BsAUG3 and BsORF31uc, uORFs dissociate from the mRNA (26). Whereas in RSV, after and Fig. 3: SeB). For this reason, we postulate that translation of translation of uORF3, ribosomes can still resume scanning to uORF3 is a prerequisite for packaging by the disruption of an reinitiate at AUGgag (Fig. 1 BsWT). inhibitory secondary structure, thus allowing binding of the In Rous sarcoma virus RNA, the distance between uORFl and Pr76gag to die \j/ region. This model could explain the uORF3 (134 nt) is well conserved between the different conservation of uORFs in die RSV leader rich in secondary avian/leukosis retrovirus strains (7,35). The conservation of the structure and diat efficient translation initiation of tiiese upstream intercistronic distance is consistent with our reinitiation model. AUGs supports a function in melting structured RNA (7,18). Indeed, as observed with deletion mutants (13 and 44 nt deletions), the intercistronic interval in RSV leader must be kept constant for efficient reinitiation at AUG3 and hence for ACKNOWLEDGEMENTS translational regulation at AUGgag. This agrees with other studies on reinitiation, where it was reported that the inhibitory We thank Andrew Craig, Greg Cosentino, Sylvie Mader and effect of inserting a single uORF on translation of downstream Robert Frederickson for critical reading of die manuscript We are product decreases as the uORF is moved further upstream (27). grateful to Nicholas Roggli for drawings and photographs and to To explain this observation, it was suggested that ribosomes Daniele Rifat for syntiiesis of oligonucleotides. One of us (OD) scanning from the uORF's termination codon to a downstream gratefully acknowledges the financial support of the Sandoz start codon require a certain period of time to bind a new set of Stiftung, Basel, Switzerland. This work was supported by grant initiation factors (34). In a mammalian system, 79 nt was found 31.29983.90 from the Swiss National Science foundation. Downloaded from https://academic.oup.com/nar/article-abstract/23/5/861/2376433 by DeepDyve user on 06 August 2020 868 Nucleic Acids Research, 1995, Vol. 23, No. 5 20 Meric, C. and SpahrP.F. (1986) J. ViroL, 60, 450-459. REFERENCES 21 De Wet, J.R., Wood, K.W., DeLuca, M., Helinski, D.R. and Subramani, S. (1987) Mol. Cell. BioL, 7, 725-737. 1 Kozak, M. (1989) J. Cell. BioL, 108, 229-241. 22 Oertle, S. and Spahr, P.F. (1990) J. Virol., 64, 5757-5763. 2 Merrick, W.C. (1992) Microbiol Rev., 56, 291-315. 23 Lo, K. M., Jones, S.S., Hackett, N. and Khorana, H.G. (1984) Proc. Natl. 3 Damiani, R.D. and Wessler, S.R. (1993) Prvc. Natl. Acad. ScL USA, 90, Acad. Sci. USA., 81, 2285-2289. 8244-8248. 24 Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492. 4 Degnin, C.R., Schleiss, M.R., Cao J. and Geballe, AP. (1993) J. ViroL. 67, 25 Hinnebusch, A.G. (1988) Proc. Natl. Acad Sci. USA, 85, 7279-7283. 5514-5521. 26 Abastado, J.P., Miller, P.F., Jackson, B.M. and Hinnebusch A.G. (1991) 5 Hill, J.R and Morris, D.R. (1993) J. BioL Chem., 268, 726-731. Mol. Cell. BioL, 11, 486-496. 6 Werner, M., Feller, A., Messenguy, F. and Pierard, A. (1987) Cell, 49, 27 Futterer, J. and Hohn, T. (1992) Nucleic Acids Res., 20, 3851-3857. 805-813. 28 Peabody, D.S. and Berg, P. (1986) Mot. Cell. BioL, 6, 2695-2703. 7 Hackett, P.B., Dalton, M.W., Johnson, D.P. and Petersen, R. (1992) Nucleic 29 Moustakas, A., Sonstegard, T.S. and Hackett, P.B. (1993) J. ViroL, 67, Acids Res., 19, 6929-6934. 4350-4357. 8 Linial, M. and MillerAD. (1990) Curr Topics Microbiol. Immunol., 157, 30 Kozak. M. (1986) Proc. Natl. Acad. Sci. USA, 83, 2850-2854. 125-152. 31 Kozak, M. (1991) J. Cell. BioL, 115, 887-903. 9 Kate, R.A., Terry, R.W. and Skalka, A.M. (1986) J. ViroL, 59, 163-167. 32 Pelletier, J. and Sonenberg, N. (1985) Cell, 40, 515-526. 10 Aronoff, R., Hajjar, AM. and Linial, M.L. (1993) J. Virol., 67, 178-188. 33 Pelletier, J. and Sonenberg, N. (1988) Nature, 334, 320-325. 11 Darlix, J.L., Spahr, PR, Bromley, P.A. and Jaton, J.C. (1979) / . Virol., 29, 34 Kozak, M. (1987) Mol. Cell. BioL, 7, 3438-3445. 597-611. 35 Bizub, D., Katz, R. A. and Skalka, A.M. (1984) / Virol, 49, 557-565. 12 Petersen, R. B., Hensel, CM . and Hackett, P.B. (1984) J. Virol., 51, 36 Cobrinik, D., Sosky, L. and Leis, J. (1988) J. ViroL, 62, 3622-3630. 727-729. 37 Cobrinik, D., Aiyar, A., Ge, Z., Katzman, M., Huang, H. and Leis, J. 13 Petersen, R. B. and Hackett, P.B. (1985) J. Virol, 56, 683-690. (1991) J. ViroL, 65, 3864-3872. 14 Hackett, P. B., Petersen, R. B. , Hensel, C. H. , Albericio, F., Gunderson, S. 38 Knight, J.B., Si, Z.H and Stoltefus, CM . (1994) J. Virol, 68, 4493-4502. I., Palmenberg, A. C. and Barany, G. (1986) J. Mol. BioL, 190, 45-57. 39 Kawai, S and T. Koyama. (1984) J. ViroL, 51, 147-153. 15 Hensel, C. H., Petersen, R. B. and Hackett, P.B. (1989) / . ViroL, 63, 40 Futterer, J. and Hohn, T. (1991) EMBO J., 10, 3887-3896. 4986-4990. 41 Hughes, S., Mellstrom, K., Kosik, E., Tamanoi, F. and Brugge, J. (1984) 16 Moustakas, A., Sonstegard, T.S. and Hackett, P.B. (1993) J. ViroL, 67, Mol. Cell BioL, 4, 1738-1746. 4337-4349. 42 Grant, CM . and Hinnebusch, A.G. (1994) Mol. Cell. BioL, 14, 606-618. 17 Petersen, R. B., Moustakas, A. and Hackett, P.B. (1989) J. ViroL, 63, 43 Miller, P. F. and Hinnebusch, A.G. (1989) Genes Dev., 3, 1217-1225. 4787^796 . 44 Mueller, P.F, Jackson, B.M., Miller, P.F. and Hinnebusch, A.G. (1988) 18 Donze", O. and Spahr, P.F. (1992) EMBO J., 11, 3747-3757. Mol. CelL BioL, 8, 5439-5447. 19 Sambrook, J., Fristch E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor University Press, Cold Spring Harbor, NY.

Journal

Nucleic Acids Research – Oxford University Press

Published: Mar 11, 1995

There are no references for this article.