Inhibition of splicing by serine-arginine rich protein 55 (SRp55) causes the appearance of partially spliced HIV-1 mRNAs in the cytoplasm.
ABSTRACT We have previously shown that SRp55 inhibits splicing from HIV-1 exon 3, thereby generating partially spliced mRNAs encoding HIV-1 vpr. Here we show that SRp55 also inhibits splicing from HIV-1 exon 5 to generate HIV-1 vpu/env mRNA, albeit with lower efficiency. We also show that inhibition of HIV-1 splicing by SRp55 causes the appearance of partially spliced vpu, env and vpr mRNAs in the cytoplasm. SRp55 could also induce production of extracellular p24gag from a rev-defective HIV-1 provirus. These results indicate that SRp55 aids in the generation of partially spliced and unspliced HIV-1 mRNAs.
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ABSTRACT: The human immunodeficiency virus type 1 (HIV-1) genome contains 20 exons that are alternatively spliced from 16 splice sites to generate more than 40 different mRNAs, including incompletely spliced and unspliced mRNAs. In contrast to avian retroviral RNA, which has a cis-acting element in gag that negatively regulates splicing (NRS), HIV-1 RNA did not have any NRS sequences in the gag or pol genes detectable by a splicing inhibition assay. However, this assay demonstrated that the HIV-1 first 5' splice site competed with a cellular 5' splice site, suggesting that HIV-1 may have some strong splice sites. To extend this observation, we used a splice site swapping strategy to determine the efficiency of 14 HIV-1 splice sites in human beta globin chimeras tested in transient transfection experiments. While the 1st HIV-1 5' splice site used in all spliced transcripts and the 4th 5' splice site used in most of the 2-kb transcripts were efficient, the other splice sites, including all the 3' splice sites, were less efficient, ranging in use from 25 to 60%. We propose that this range of splice site efficiencies contributes to the regulation of alternative splicing of HIV-1 mRNAs.Virology 12/1995; 213(2):373-85. · 3.37 Impact Factor
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ABSTRACT: SR proteins have a characteristic C-terminal Ser/Arg-rich repeat (RS domain) of variable length and constitute a family of highly conserved nuclear phosphoproteins that can function as both essential and alternative pre-mRNA splicing factors. We have cloned a cDNA encoding a novel human SR protein designated SRp30c, which has an unusually short RS domain. We also cloned cDNAs encoding the human homologues of Drosophila SRp55/B52 and rat SRp40/HRS. Recombinant proteins expressed from these cDNAs are active in constitutive splicing, as shown by their ability to complement a HeLa cell S100 extract deficient in SR proteins. Additional cDNA clones reflect extensive alternative splicing of SRp40 and SRp55 pre-mRNAs. The predicted protein isoforms lack the C-terminal RS domain and might be involved in feedback regulatory loops. The ability of human SRp30c, SRp40 and SRp55 to modulate alternative splicing in vivo was compared with that of other SR proteins using a transient contransfection assay. The overexpression of individual SR proteins in HeLa cells affected the choice of alternative 5' splice sites of adenovirus E1A and/or human beta-thalassemia reporters. The resulting splicing patterns were characteristic for each SR protein. Consistent with the postulated importance of SR proteins in alternative splicing in vivo, we demonstrate complex changes in the levels of mRNAs encoding the above SR proteins upon T cell activation, concomitant with changes in the expression of alternatively spliced isoforms of CD44 and CD45.The EMBO Journal 10/1995; 14(17):4336-49. · 9.82 Impact Factor
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ABSTRACT: The requirement of human immunodeficiency virus type 1 to generate numerous proteins from a single primary transcript is met largely by the use of suboptimal splicing to generate over 30 mRNAs. To ensure that appropriate quantities of each protein are produced, there must be a signal(s) that controls the efficiency with which any particular splice site in the RNA is used. To identify this control element(s) and to understand how it operates to generate the splicing pattern observed, we have initially focused on the control of splicing of the tat-rev intron, which spans the majority of the env open reading frame. Previous analysis indicated that a suboptimal branchpoint and polypyridimine tract in this intron contribute to its suboptimal splicing (A. Staffa and A. Cochrane, J. Virol. 68:3071-3079, 1994). In this report, we identify two additional elements within the 3'-terminal exon, an exon-splicing enhancer (ESE) and an exon splicing silencer (ESS), that modulate the overall efficiency with which the 3' tat-rev splice site is utilized. Both elements are capable of functioning independently of one another. Furthermore, while both the ESE and ESS can function in a heterologous context, the function of the ESS is extremely sensitive to the sequence context into which it is placed. In conclusion, it would appear that the presence of a suboptimal branchpoint and a polypyrimidine tract as well as the ESE and ESS operate together to yield the balanced splicing of the tat-rev intron observed in vivo.Molecular and Cellular Biology 09/1995; 15(8):4597-605. · 5.37 Impact Factor