Differential Effects of the SR Proteins 9G8, SC35, ASF/SF2, and SRp40 on the Utilization of the A1 to A5 Splicing Sites of HIV-1 RNA

Ropers, Delphine; Ayadi, Lilia; Gattoni, Renata; Jacquenet, S.; Damier, Laurence; Branlant, Christiane; Stévenin, James

doi:10.1074/jbc.m404452200

Cited by 70 publications

(110 citation statements)

References 65 publications

(100 reference statements)

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“…Indeed, to express key viral proteins, HIV-1 uses a combination of several alternative 5Ј and 3Ј splice sites to generate Ͼ40 different mRNAs from its singly transcribed genomic premRNA. The choice of these alternative splice sites is strongly influenced and regulated by interactions of specific HIV premRNA sequences with SR proteins (30). The HIV-1 transcript therefore constitutes an ideal natural substrate to test the efficacy of our drugs on alternative splicing.…”

Section: Resultsmentioning

confidence: 99%

Selective modification of alternative splicing by indole derivatives that target serine-arginine-rich protein splicing factors

Soret

Bakkour

Maire

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

111

106

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The prevalence of alternative splicing as a target for alterations leading to human genetic disorders makes it highly relevant for therapy. Here we have used in vitro splicing reactions with different splicing reporter constructs to screen 4,000 chemical compounds for their ability to selectively inhibit spliceosome assembly and splicing. We discovered indole derivatives as potent inhibitors of the splicing reaction. Importantly, compounds of this family specifically inhibit exonic splicing enhancer (ESE)-dependent splicing, because they interact directly and selectively with members of the serine-arginine-rich protein family. Treatment of cells expressing reporter constructs with ESE sequences demonstrated that selected indole derivatives mediate inhibition of ESE usage in vivo and prevent early splicing events required for HIV replication. This discovery opens the exciting possibility of a causal pharmacological treatment of aberrant splicing in human genetic disorders and development of new antiviral therapeutic approaches.splicing correction ͉ exonic splicing enhancer ͉ small chemicals ͉ pathologic splicing R emoval of introns from newly transcribed RNA polymerase II precursors (pre-mRNA) during splicing not only is an essential step for the expression of most genes in higher eukaryotic cells but also constitutes an important mechanism for generation of protein diversity and regulation of gene expression (1, 2). It is estimated that Ͼ70% of human genes are subjected to alternative splicing, and it is not surprising that many point mutations causing human diseases are associated with aberrant splicing (3, 4).Current models of constitutive and a fortiori alternative splicing suggest that splice site recognition is strongly modulated by the interaction of specific exonic and intronic pre-mRNA sequences with at least two classes of nonspliceosomal nuclear RNA-binding proteins: serine-arginine-rich (SR) proteins (5-7) and heterogeneous nuclear ribonucleoproteins (8-10). These proteins interact with spliceosomal components (5-7) and either activate or prevent the use of degenerate splice sites in their vicinity. Thus, binding of SR proteins to exonic splicing enhancers (ESE) through their RNA-recognition motif (RRM) promotes exon definition by recruiting constitutive factors via protein-protein interactions mediated by their arginine-serine-rich (RS) domain and prevents the action of nearby splicing silencers (4, 6, 11).Mutations causing human diseases may affect splice sites as well as regulatory sequences leading to the production of defective proteins (4, 11). Thus, targeting either the mutated sequences or the factors that bind them may prove to be a valuable strategy to correct aberrant splicing. Recently, antisense strategies targeting ESEdependent mechanisms have been used to induce skipping of exons containing nonsense mutations or, conversely, to restore exon inclusion by synthetic exon-specific effectors (bifunctional antisense peptide molecules or tailed antisense oligonucleotides) or spliceosome-mediat...

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Section: Resultsmentioning

confidence: 99%

Selective modification of alternative splicing by indole derivatives that target serine-arginine-rich protein splicing factors

Soret

Bakkour

Maire

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

111

106

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“…1A) splicing to exons 4a and 5 (25,26). In contrast, overexpression of ASF/SF2 promotes production of the partially spliced vpr and overrides the function of GAR, much like SRp55 (39,40). Because both ASF/SF2 and SRp55 can bind to exon 5, one may speculate that these interactions in combination with other binding sites only occupied in the presence of high levels of ASF/SF2 or SRp55, may inhibit splicing from exon 3 to exons 4, 4a, 4b, 4c, and 5 thereby creating the vpr mRNA.…”

Section: Discussionmentioning

confidence: 99%

Serine- and Arginine-rich Proteins 55 and 75 (SRp55 and SRp75) Induce Production of HIV-1 vpr mRNA by Inhibiting the 5′-Splice Site of Exon 3

Tranell

Fenyõ

Schwartz

2010

Journal of Biological Chemistry

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HIV-1 non-coding exon 3 can either be spliced to exons 4, 4a, 4b, 4c, and 5 to generate tat, rev, and nef mRNAs or remain unspliced to produce the 13a7 vpr mRNA. Here we show that serine-and arginine-rich proteins 55 and 75 (SRp55 and SRp75) inhibit splicing from the 5-splice site of exon 3 thereby causing an accumulation of the partially unspliced 13a7 vpr mRNA. In contrast, serineand arginine-rich protein 40 (SRp40) induces splicing from exon 3 to exon 4, thereby promoting the production of the 1347 tat mRNA. We demonstrate that SRp55 stimulates vpr mRNA production by interacting with the previously identified HIV-1 splicing enhancer named GAR and inhibiting its function. This inhibition requires both serine arginine-rich and RNA-binding domains of SRp55, indicating that production of HIV-1 vpr mRNA depends on the interaction of SRp55 with an unknown factor.To produce all the mRNAs that are needed for HIV-1 to be infectious it uses alternative splicing. The full-length 9-kb transcript contains several splice sites, including four 5Ј-splice sites, also called splice donors (SD1-4) 2 and eight 3Ј-splice sites, also called splice acceptors (SA2-3, -4a-c, -5, and -7) (supplemental Fig. 1A), which can all be used in different combinations to create more than 35 differently spliced mRNAs (supplemental Fig. 1, B and C) (1-6). Cloning and expression of individual HIV-1 mRNAs have revealed that mRNAs spliced to SA3 produce Vpr, those spliced to SA4 produce Tat, those spliced to SA4a and -4b produce Rev and Nef, and those spliced to SA5 produce Nef (1,2,7,8). mRNAs spliced to SA2 are believed to produce Vif (5, 7). Two additional mRNAs that are believed to produce Rev and Nef are spliced to SA4c or to SA7, respectively (4). To produce the correct amounts of all mRNAs, the splice acceptors in HIV-1 are sub-optimal because of short and interrupted polypyrimidine tracts, non-canonical branch points, and inhibitory sequences that down-regulate the usage of several splice sites (6, 9 -13).Enhancer or silencer sequences regulate alternative splicing by up-regulating or down-regulating the usage of a splice site (14). Generally, the family of serine-and arginine-rich proteins (SR proteins) target enhancer sequences, and silencer sequences are targeted by heterogeneous nuclear ribonucleoproteins (15). There are several enhancers and silencers on HIV-1 pre-mRNA (16). A silencer in exon 3 called ESSV inhibits the vpr splice acceptor SA3 (10, 17). Two silencers in exon 4, named ESS2 and ESS2p, inhibit splicing into exon 4 thereby inhibiting the production of tat mRNA (18,19). SA7 is used by all mRNAs in the 2-kb class. In exon 7 a silencer called ESS3 blocks U2 small nuclear ribonucleoprotein, thereby suppressing SA7 (19 -21). An intronic silencer that blocks SA7 is located in the intron, directly upstream of exon 7 (22). An enhancer element called ESE3 that activates SA7 is located close to ESS3 in exon 7 (9, 21, 23). An enhancer in exon 4 has been named ESE2, because it attenuates ESS2 and thereby enhances splicing into SA4 (24)...

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“…Alternative splicing plays a key role in the generation of viral proteins during infection, and ASF/SF2 has been shown to regulate pre-mRNA splicing of HIV-1 (35). Such hijacking of host splicing factors by viruses may result in aberrant expression and/or modification of these proteins.…”

Section: Discussionmentioning

confidence: 99%

Alternative Splicing Factor/Splicing Factor 2 Regulates the Expression of the ζ Subunit of the Human T Cell Receptor-associated CD3 Complex

Moulton

Tsokos

2010

Journal of Biological Chemistry

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T cells from patients with systemic lupus erythematosus express decreased levels of the T cell receptor-associated CD3 chain, a feature directly linked to their aberrant function. The decrease in CD3 protein expression is in part due to decreased levels of functional wild type isoform of the 3-untranslated region (UTR) of CD3 mRNA with concomitant increased levels of an unstable alternatively spliced isoform. In order to identify factors involved in the post-transcriptional regulation of CD3, we performed mass spectrometric analysis of Jurkat T cell nuclear proteins "pulled down" by a CD3 3-UTR oligonucleotide, which identified the splicing protein alternative splicing factor/splicing factor 2 (ASF/SF2). We show for the first time that ASF/SF2 binds specifically to the 3-UTR of CD3 and regulates expression of CD3 protein by limiting the production of the alternatively spliced isoform. During activation of human T cells, an increase in the wild type CD3 mRNA is associated with increased expression of ASF/SF2. Finally, we show a significant correlation between ASF/SF2 and CD3 protein levels in T cells from systemic lupus erythematosus patients. Thus, our results identify ASF/SF2 as a novel factor in the regulation of alternative splicing of the 3-UTR of CD3 and protein expression in human T cells. Systemic lupus erythematosus (SLE)2 is a chronic multisystem autoimmune disease characterized by abnormal cellular and humoral immune responses and a disease course marked by relapses (flares) and remissions. A key signaling defect of T cells in SLE patients is their aberrant expression of the T cell receptor (TCR)-associated CD3 chain, the crucial signaling transducer of the TCR (1). CD3 protein expression is heterogeneous in SLE patients and tends to inversely correlate with severity of the disease. Consequently, many patients exhibit decreased (20 -80% of normal) levels of CD3 protein, whereas others express normal levels of CD3 protein when compared with healthy individuals. The homologous Fc receptor ␥ chain replaces CD3 chain in the TCR CD3 complex in these CD3-low T cells (2) and recruits the spleen tyrosine kinase (Syk) instead of the normally recruited -associated protein (ZAP-70) (3). This "rewiring" of the TCR, along with preclustered TCRbearing lipid rafts, leads to aberrantly increased phosphorylation and calcium flux upon activation (4). Despite this overexcitable phenotype, SLE T cells fail to produce the vital cytokine interleukin-2. Replenishment of the CD3 in the SLE T cells in vitro reverts this phenotype and more importantly restores interleukin-2 production (5). Although these findings highlight a central role for the aberrant CD3 expression in the SLE T cell defect, the mechanisms regulating the expression of CD3 chain in physiology and in disease are not fully understood.The CD3 gene spans 31 kb in the chromosome 1q23.1 locus, a region associated with SLE susceptibility (6), and bears eight exons separated by introns ranging from 700 bp to greater than 8 kb (Fig. 1A, top) (7,8). The CD3 mRNA is a 1...

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Differential Effects of the SR Proteins 9G8, SC35, ASF/SF2, and SRp40 on the Utilization of the A1 to A5 Splicing Sites of HIV-1 RNA

Cited by 70 publications

References 65 publications

Selective modification of alternative splicing by indole derivatives that target serine-arginine-rich protein splicing factors

Selective modification of alternative splicing by indole derivatives that target serine-arginine-rich protein splicing factors

Serine- and Arginine-rich Proteins 55 and 75 (SRp55 and SRp75) Induce Production of HIV-1 vpr mRNA by Inhibiting the 5′-Splice Site of Exon 3

Alternative Splicing Factor/Splicing Factor 2 Regulates the Expression of the ζ Subunit of the Human T Cell Receptor-associated CD3 Complex

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