Overlapping Cis Sites Used for Splicing of HIV-1env/nefand revmRNAs

Abstract

Alternative splicing is used to generate more than 30 human immunodeficiency virus type 1 (HIV-1) spliced and unspliced mRNAs from a single primary transcript. The abundance of HIV-1 mRNAs is determined by the efficiencies with which its different 5′ and 3′ splice sites are used. Three splice sites (A4c, A4a, and A4b) are upstream of the rev initiator AUG. RNAs spliced at A4c, A4a, and A4b are used as mRNAs for Rev. Another 3′ splice site (A5) is immediately downstream of the revinitiator. RNAs spliced at A5 are used as mRNAs for Env and Nef. In this report, primer extension analysis of splicing intermediates was used to show that there are eight branch points in this region, all of which map to adenosine residues. In addition, cis elements recognized by the cellular splicing machinery overlap; the two most 3′ branch points overlap with the AG dinucleotides at rev 3′ splice sites A4a and A4b. Competition of the overlapping cis sites for different splicing factors may play a role in maintaining the appropriate balance of mRNAs in HIV-1-infected cells. In support of this possibility, mutations at rev 3′ splice site A4b AG dinucleotide dramatically increased splicing of the env/nef3′ splice site A5. This correlated with increased usage of the four most 3′ branch points, which include those within the rev3′ splice site AG dinucleotides. Consistent with these results, analysis of a mutant in which three of the four env/nefbranch points were inactivated indicated that use of splice site A5 was inhibited and splicing was shifted predominantly to the most 5′rev 3′ splice site A4c with preferential use of the two most 5′ branch points. Our results suggest that spliceosomes formed atrev A4a-4b, rev A4c, and env/nef A5 3′ splice sites each recognize different subsets of the eight branch point sequences. Alternative splicing is used to generate more than 30 human immunodeficiency virus type 1 (HIV-1) spliced and unspliced mRNAs from a single primary transcript. The abundance of HIV-1 mRNAs is determined by the efficiencies with which its different 5′ and 3′ splice sites are used. Three splice sites (A4c, A4a, and A4b) are upstream of the rev initiator AUG. RNAs spliced at A4c, A4a, and A4b are used as mRNAs for Rev. Another 3′ splice site (A5) is immediately downstream of the revinitiator. RNAs spliced at A5 are used as mRNAs for Env and Nef. In this report, primer extension analysis of splicing intermediates was used to show that there are eight branch points in this region, all of which map to adenosine residues. In addition, cis elements recognized by the cellular splicing machinery overlap; the two most 3′ branch points overlap with the AG dinucleotides at rev 3′ splice sites A4a and A4b. Competition of the overlapping cis sites for different splicing factors may play a role in maintaining the appropriate balance of mRNAs in HIV-1-infected cells. In support of this possibility, mutations at rev 3′ splice site A4b AG dinucleotide dramatically increased splicing of the env/nef3′ splice site A5. This correlated with increased usage of the four most 3′ branch points, which include those within the rev3′ splice site AG dinucleotides. Consistent with these results, analysis of a mutant in which three of the four env/nefbranch points were inactivated indicated that use of splice site A5 was inhibited and splicing was shifted predominantly to the most 5′rev 3′ splice site A4c with preferential use of the two most 5′ branch points. Our results suggest that spliceosomes formed atrev A4a-4b, rev A4c, and env/nef A5 3′ splice sites each recognize different subsets of the eight branch point sequences. small nuclear ribonucleoprotein nucleotide(s) human immunodeficiency virus type 1 exon-splicing silencer element 1,4-piperazinediethanesulfonic acid. Splicing of mRNA precursors in the nucleus of metazoan cells occurs by the cleavage and joining of 5′ and 3′ splice sites. This is mediated by recognition of conserved cis-acting sequences near the two splice sites that removes the intron between these splice sites. The splicing process is catalyzed by the cellular splicing machinery within spliceosome complexes (for reviews, see Refs. 1Moore M.J. Query C.C. Sharp P.A. Gesteland R.F. Atkins J.R. The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 303-357Google Scholar, 2Kramer A. Annu. Rev. Biochem. 1996; 65: 367-409Crossref PubMed Scopus (620) Google Scholar, 3Staley J.P. Guthrie C. Cell. 1998; 92: 315-326Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar). The assembly of spliceosomes involves an initial interaction with U1 snRNP1 at the 5′ splice site. The 3′ splice site is then recognized by factor U2AF binding to a greater than 12-nt polypyrimidine tract upstream of the AG dinucleotide, which borders the site of 3′ cleavage. Another cis element, the branch point sequence, is located 18–40 nt upstream of the AG dinucleotide with the loosely conserved consensus sequence YNYURAY (the underlined residue is the branch point). The branch point sequence is recognized by U2 snRNP, whose binding is facilitated by the protein U2AF. This step is followed by binding of U5 and U4/U6 snRNPs as a triple snRNP to form the complete spliceosome. The splicing process for most cellular mRNA precursors is normally efficient. However, processing of some pre-mRNAs occurs by tissue-specific or developmentally regulated alternative splicing pathways, resulting in multiple mRNAs produced for a single pre-mRNA. This involves the use of alternative 5′ or 3′ splice sites, exon skipping, intron inclusion, and mutually exclusive exons. Such regulated splicing results in the production of different proteins from the same gene and is an important mechanism for expanding the repertoire of cellular gene expression (1Moore M.J. Query C.C. Sharp P.A. Gesteland R.F. Atkins J.R. The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 303-357Google Scholar). HIV-1 uses the cellular splicing machinery to express its genes. Over 30 different HIV-1 mRNAs are derived by alternative splicing from a single RNA precursor. In addition, approximately half of the RNA transcripts remain unspliced. These primary transcripts are packaged into progeny virions and are used as mRNA for the gagand pol gene products. It has been shown that HIV-1 RNA splicing efficiency is primarily determined by the relative strengths of the various 3′ splice sites in the viral RNA (4O'Reilly M.M. McNally M.T. Beemon K.L. Virology. 1995; 213: 373-385Crossref PubMed Scopus (101) Google Scholar, 5Staffa A. Cochrane A. J. Virol. 1994; 68: 3071-3079Crossref PubMed Google Scholar, 6Si Z.-H. Amendt B.A. Stoltzfus C.M. Nucleic Acids Res. 1997; 25: 861-867Crossref PubMed Scopus (86) Google Scholar). Several cis elements have been shown to affect HIV-1 3′ splice site usage. These include suboptimal splice sites (polypyrimidine tracts and branch point sequences) as well as enhancer and silencer elements mapping within the exons downstream of the splice sites (4O'Reilly M.M. McNally M.T. Beemon K.L. Virology. 1995; 213: 373-385Crossref PubMed Scopus (101) Google Scholar, 5Staffa A. Cochrane A. J. Virol. 1994; 68: 3071-3079Crossref PubMed Google Scholar, 6Si Z.-H. Amendt B.A. Stoltzfus C.M. Nucleic Acids Res. 1997; 25: 861-867Crossref PubMed Scopus (86) Google Scholar, 7Wentz M.P. Moore B.E. Cloyd M.W. Berget S.M. Donehower L.A. J. Virol. 1997; 71: 8542-8551Crossref PubMed Google Scholar, 8Staffa A. Cochrane A. Mol. Cell. Biol. 1995; 15: 4597-4605Crossref PubMed Scopus (152) Google Scholar, 9Amendt B.A. Hesslein D. Chang L.-J. Stoltzfus C.M. Mol. Cell. Biol. 1994; 14: 3960-3970Crossref PubMed Scopus (125) Google Scholar, 10Amendt B.A. Si Z.-H. Stoltzfus C.M. Mol. Cell. Biol. 1995; 15: 4606-4615Crossref PubMed Scopus (123) Google Scholar, 11Dyhr-Mikkelsen H. Kjems J. J. Biol. Chem. 1995; 270: 24060-24066Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 12Damier L. Domenjoud L. Brantlant C. Biochem. Biophys. Res. Commun. 1997; 237: 182-187Crossref PubMed Scopus (22) Google Scholar). The levels of tat, rev, nef, andenv mRNAs in HIV-1-infected cells are determined in part by splicing. Several alternative 3′ splice sites are present in a 300-nt region in the middle of the HIV genome (splice sites A3, A4a, A4b, A4c, and A5; Fig. 1). In addition, a 5′ splice site (D4) downstream of these alternative 3′ splice sites is spliced to 3′ splice site A7 within the env coding sequence (Fig. 1). When both splicing events occur, multiply spliced mRNAs are created.tat mRNAs are spliced at site A3, whereas revmRNAs are spliced at either A4a, A4b, or A4c. Most envand nef mRNAs are spliced at site A5; however,env mRNA remains single-spliced (13Purcell D.F.J. Martin M.A. J. Virol. 1993; 67: 6365-6378Crossref PubMed Google Scholar). Splicing between sites D4 and A7 creates a noncoding exon upstream of the nefreading frame. In addition to alternative splicing, the levels of single-spliced and unspliced RNA are also selectively regulated by the HIV-1 Rev protein (for a review see Ref. 14Cullen B.R. Annu. Rev. Microbiol. 1991; 45: 219-250Crossref PubMed Scopus (114) Google Scholar). This small basic protein binds to an RNA element in the env gene called the Rev-responsive element within the env gene and facilitates transport of the unspliced and single-spliced RNA from the nucleus to the cytoplasm. We have previously shown that the efficiency of splicing at splice site A3, which is primarily used for synthesis of tat mRNA, is determined by both a suboptimal polypyrimidine tract and by an exon-splicing silencer element (ESS) (6Si Z.-H. Amendt B.A. Stoltzfus C.M. Nucleic Acids Res. 1997; 25: 861-867Crossref PubMed Scopus (86) Google Scholar). However, the ESS, which maps approximately 60 nt downstream of the tat splice site, does not significantly inhibit splicing at the rev andenv splice sites A4a, A4b, A4c, and A5, which are downstream of the ESS (10Amendt B.A. Si Z.-H. Stoltzfus C.M. Mol. Cell. Biol. 1995; 15: 4606-4615Crossref PubMed Scopus (123) Google Scholar). Splicing at these latter closely spaced 3′ splice sites, which are utilized for approximately 90% of the spliced mRNAs in HIV-1-infected cells (13Purcell D.F.J. Martin M.A. J. Virol. 1993; 67: 6365-6378Crossref PubMed Google Scholar), may involve the assembly of a single spliceosome complex upstream of the A4c splice site followed by scanning to the four different AGs. The relative usage of the different splice sites would then be determined by competition between the AGs based on their position and sequence context (15Smith C.W.J. Porro E.B. Patton J.G. Nadal-Ginard B. Nature. 1989; 342: 243-247Crossref PubMed Scopus (208) Google Scholar, 16Smith C.W.J. Chu T.T. Nadal-Ginard B. Mol. Cell. Biol. 1993; 13: 4939-4952Crossref PubMed Scopus (170) Google Scholar). This predicts that only one branch point sequence or one set of branch point sequences would be recognized by the U2 snRNP. Alternatively, separate spliceosomes may form upstream of each AG. This model predicts that different branch points or sets of branch points corresponding to different binding sites for U2 snRNP would be used for splicing at each of these AGs. To distinguish these models, we identified branch points in the 80-nt region containing the four 3′ splice sites by primer extension analysis. Our results indicate that separate spliceosomes form and that there is overlap of the cis splicing sequences recognized by the cellular splicing machinery. Plasmid pHS1-X (9Amendt B.A. Hesslein D. Chang L.-J. Stoltzfus C.M. Mol. Cell. Biol. 1994; 14: 3960-3970Crossref PubMed Scopus (125) Google Scholar), which contains HIV-1 sequences derived from the infectious clone pNL4–3 (GenBankTMaccession no. M19921), was used to construct plasmids as templates to synthesize RNA substrates for splicing (Fig. 1). In order to facilitate cloning, plasmid pHS1-ΔSac was constructed in which a SacI site in the vector sequence of pHS1-X was removed by blunt end ligation to create a unique SacI site at nt 5999 of the pNL4–3 sequence. Mutant plasmids 4B-S, 4B-/5-, and 5-, which inactivate splice acceptors A4b and/or A5, were created by replacing the region between the Bsu36I (nt 5954 in HIV-1) and SacI (nt 5999 in HIV-1) restriction enzyme sites with hybridized oligonucleotides. The following oligonucleotides were used (changes from wild type are underlined): mutant 4B-S (sense, 5′-TTACGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGAAGAGCT-3′; antisense, 5′-CTTCGTCGCTGTCTCCGCTTCTTCCTGCCATAGGAGATGCG-3′); mutant 4B-/5- (sense, 5′-TTACGCATCTCCTATGGCCGTAAGAAGCGGAGACAGCGACGAAGAGCT-3′; antisense, 5′-CTTCGTCGCTGTCTCCGCTTCTTACGGCCATAGGAGATGCG-3′); mutant 5- (sense, 5′-TTAGGCATCTCCTATGGCCGTAAGAAGCGGAGACAGCGACGAAGAGCT-3′; antisense, 5′-CTTCGTCGCTGTCTCCGCTTCTTACGGCCATAGGAGATGCC-3′). pNL4–3 plasmids with mutations in splice acceptor A4b (pNLΔSA4b (13Purcell D.F.J. Martin M.A. J. Virol. 1993; 67: 6365-6378Crossref PubMed Google Scholar) and p4BSAM (17Riggs N.L. Virology. 1994; PubMed Scopus Google were by D. for and J. of and containing mutations and were constructed by to SacI of p4BSAM and to of to the appropriate vector derived from Plasmid which branch points and was created with primer and primer and used to the by The resulting 300-nt was and SacI and with the restriction of was used to construct mutant plasmid to in 3′ splice site A4b) with primer and primer to the The in the plasmids used as templates for splicing were by Plasmid containing human downstream of an RNA has been previously B. Cell. Full Text PDF PubMed Scopus Google Scholar). In RNA transcripts with were as (9Amendt B.A. Hesslein D. Chang L.-J. Stoltzfus C.M. Mol. Cell. Biol. 1994; 14: 3960-3970Crossref PubMed Scopus (125) Google Scholar). To pHS1-ΔSac and its were and was In were in a containing of of of or RNA and approximately 1 of Splicing were as previously (9Amendt B.A. Hesslein D. Chang L.-J. Stoltzfus C.M. Mol. Cell. Biol. 1994; 14: 3960-3970Crossref PubMed Scopus (125) Google Scholar). In a of nuclear in Nucleic Acids Res. PubMed Scopus Google Scholar), and was with approximately of RNA for at 30 In order to the of for primer extension the splicing was at 1 and approximately of RNA was used. RNA splicing were on for at extension analysis was as previously J. Laboratory Cold Spring Harbor Cold Spring Harbor, Scholar). and unspliced RNA were from the in and in a containing and at pHS1-ΔSac and primer at a of 1 was in a containing of and 30 of was by was hybridized to RNA at 30 in a containing 1 extension to the branch point sites was in a containing a of each and of virus was also used for The were on a for at of corresponding to the splice site region the primer was on the in order to map the of the branch points. extension analysis of was the that primer was used of primer We primer extension analysis to branch points in the 80-nt region containing the and env 3′ splice sites A4b, A4c, and The of a splicing of an RNA from pHS1-ΔSac were on a intermediates were and a to the exon was used to a to the branch points virus a for the of the we RNA to the same and used as for a we a primer extension analysis to the branch point site used in the splicing of exons 1 and of derived from the human gene 1 and based on results B. Cell. Full Text PDF PubMed Scopus Google Scholar), the branch point to an adenosine within the sequence upstream from the 3′ splice site. In the of the HIV-1 corresponding to eight branch points were and all of these branch points were to adenosine 1 of the sequences containing these branch points indicated that there was a to the consensus branch point sequence from three of to of the most 3′ branch point point to the adenosine residue within the AG dinucleotide of the rev A4b 3′ splice site. In addition, a of at the rev A4a 3′ splice site point point sequences in the region of HIV-1 rev and env/nef 3′ splice in sequence are shown on the and of are shown in in sequence are shown on the and of are shown in in a The that branch points and with the rev AG dinucleotides that these branch points were used in splicing at 3′ splice site A5. To this we correlated the use of the different branch points with the use of splice sites. It has been previously that mutations of the rev A4b AG dinucleotide a of virus and usage of tat and the rev 3′ splice sites (17Riggs N.L. Virology. 1994; PubMed Scopus Google Scholar). We four different mutant and Fig. as splicing at 3′ splice site A4b was with all mutant Splicing at 3′ splice site A5 of substrates with AG to and mutations was dramatically increased with wild the splicing of the AG to mutant was only with wild In each there was a in splicing at the rev A4a 3′ splice this was most for the 4B-S with the in splicing at A5 there was a in the of and intermediates to the (Fig. This to a with the AG to RNA intermediates containing would be to more on this that the branch points used for splicing at the env/nef 3′ splice site A5 are downstream than those that are used for splicing at the rev 3′ splice sites A4a, A4b, and A4c. To the of the branch points used in splicing of A4b we primer extension analysis of the mutant It be in that splicing at the A5 3′ splice site was correlated with a relative in of the four most 3′ branch point sites points in 4B-S and three of the four branch point sites points in substrates and The and in which the AG dinucleotide was to and not branch at the A4b 3′ splice site point at this site with mutant 4B-S, in which the AG dinucleotide was to the of the AG adenosine is for branch point at the A4b 3′ splice site. The in the use of branch points relative to the use of branch points correlated with increased use of 3′ splice site A5, that these branch points are used for splicing at this site. the with the mutant in which the AG dinucleotide was to the to splicing at A5 was Fig. and there was a more usage of all eight branch points. The that between the splicing to the A5 splice site of the A4b and the increased use of downstream branch points in the intermediates that the four most 3′ branch points were used for splicing to A5. To the role of the branch points identified in A5 splicing, we used in which the branch points for A5 were from adenosine to the present in the A4b and branch point with mutations of the two A5 branch points points and The branch point within the A4a AG dinucleotide was not these three branch points were for splicing to the A5 3′ splice site, we that the splicing of that of a in which A4b and env/nef A5 3′ splice sites were inactivated shown in the splicing of 4B-/5- was to that of mutant substrates and 4B-/5- both an increased of spliced at rev splice site A4c with wild shown in Fig. for this correlated with a relative in the usage of branch points 1 and This that these branch points may be used for splicing at A4c. was also a of splicing at 3′ splice site A5 (Fig. and this was correlated with a small of at the rev A4a 3′ splice site point which remains in (Fig. The to increased splicing at A4c mutations of both the A4b AG dinucleotide and either the A5 AG dinucleotide or the A5 branch points. of the A5 AG dinucleotide not show the preferential use of the A4c 3′ splice site. The AG to at A4b the in splicing at A5 In this we that different sets of branch points are used for splicing at the different HIV-1 rev 3′ splice sites. This that separate spliceosome complexes used for splicing at the rev and mRNAs form upstream of the different AG dinucleotides. It that there are a of different binding sites for U2 the of the branch points, we the of the polypyrimidine tracts used for spliceosomes at the different 3′ splice sites The polypyrimidine tract is downstream of the branch points used for splicing at a site. the polypyrimidine tract used for the env/nef 3′ splice site A5 at nt is the sequence nt Consistent with branch points which are used for splicing at site A5, are all upstream of this sequence. polypyrimidine tract located between nt and is upstream of branch points and downstream of branch points 1 and branch points 1 and may be used for splicing at rev 3′ splice site A4c. This is in with the shown in Fig. an in spliced at site A4c correlated with the increased use of branch points 1 and It is that the rev 3′ splice sites A4a and A4b use a set of branch points, of the A4a AG in the context of HIV-1 has been shown to in a in splicing at the A4b splice site with or in splicing at A5 (17Riggs N.L. Virology. 1994; PubMed Scopus Google Scholar). It that a polypyrimidine tract for rev 3′ splice sites A4a and A4b is the sequence from nt to which is downstream of branch points upstream of branch points all of these polypyrimidine tracts are than the consensus than and have residues. as has been shown with HIV 3′ splice sites, the polypyrimidine tracts of the rev splice sites also to be suboptimal (4O'Reilly M.M. McNally M.T. Beemon K.L. Virology. 1995; 213: 373-385Crossref PubMed Scopus (101) Google Scholar, 5Staffa A. Cochrane A. J. Virol. 1994; 68: 3071-3079Crossref PubMed Google Scholar, 6Si Z.-H. Amendt B.A. Stoltzfus C.M. Nucleic Acids Res. 1997; 25: 861-867Crossref PubMed Scopus (86) Google Scholar). We also in this most of the branch point sequences significantly from the metazoan consensus sequence, all of the branch points in the region of the HIV-1 rev 3′ splice sites map to adenosine residues. of HIV-1 branch points that than adenosine are used. and Kjems that the branch point for the 3′ splice site A7 maps to a residue within the sequence H. Kjems J. J. Biol. Chem. 1995; 270: 24060-24066Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). the branch point for 3′ splice site to a residue within the sequence L. Domenjoud L. Brantlant C. Biochem. Biophys. Res. Commun. 1997; 237: 182-187Crossref PubMed Scopus (22) Google Scholar). Our results show that the use of branch points is not for all HIV-1 3′ splice sites. the adenosine within the AG dinucleotides of the rev 3′ splice sites A4a and A4b also both as branch points for splicing at env/nef 3′ splice site A5. To there is for overlap of these two cis elements within two alternative 3′ splice sites. We also that each of the HIV-1 splice sites to use multiple branch points. This has previously been shown for a of spliced viral and cellular mRNAs C. PubMed Scopus Google Scholar, J. Nucleic Acids Res. PubMed Scopus Google Scholar, Nucleic Acids Res. 1989; PubMed Scopus (55) Google Scholar, H. J. J. A. PubMed Scopus Google Scholar). We that substrates with different mutations of A4b AG dinucleotide in which the AG was to or in in splicing at the env/nef 3′ splice site A5, whereas a in which the AG was to does not show this these in splicing be correlated with results in which the of these mutations were in the context of HIV-1 of these to and AG to are and (17Riggs N.L. Virology. 1994; PubMed Scopus Google Scholar, J. A. PubMed Scopus Google Scholar). In the of the AG to this was shown to involve a relative in levels of mRNAs spliced at the rev 3′ splice sites as well as 3′ splice site (17Riggs N.L. Virology. 1994; PubMed Scopus Google Scholar). In the AG to mutant has wild type Rev and is (13Purcell D.F.J. Martin M.A. J. Virol. 1993; 67: 6365-6378Crossref PubMed Google Scholar). Our results suggest that the HIV-1 may be by an splicing of the viral resulting in an in splicing at the env/nef 3′ splice site A5 and a to splice at the rev 3′ splice sites. In the in the 3′ splice site A4b mutations not the same on tat splicing as in the context of virus We not this in the in the two is that in cells the alternative 3′ splice sites may for splicing in splicing efficiency at the env/nef 3′ splice site may a in splicing at the tat 3′ splice sites. In the in splicing these factors may not be splicing in and in be to this Our results suggest that the rev A4b 3′ splice site AG dinucleotide to the of HIV-1 RNA splicing by splicing at the env/nef 3′ splice site A5. The only A4b that splicing to significantly splicing at 3′ splice site A5 is to In both this mutant and the to branch point adenosine was used Fig. that the to branch within this AG dinucleotide is not correlated with of splicing at 3′ splice site A5. the use of branch point was by the to and to the of splicing at 3′ splice site A5 was the to is unique in that a AG dinucleotide is created one Several different protein factors that with the AG dinucleotide both and in spliceosome assembly have been identified by L. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar, J. 1997; PubMed Scopus Google Scholar). is that factors at or near the rev A4b 3′ splice site may with assembly of to splicing at env/nef 3′ splice site A5. Alternatively, the three A4b AG mutations that 3′ splice site A5 splicing may a in the RNA that more of spliceosomes at the A5 splice site. We and for and on the