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Insights into the selective activation of alternatively used splice acceptors by the human immunodeficiency virus type-1 bidirectional splicing enhancer.

Asang C, Hauber I, Schaal H - Nucleic Acids Res. (2008)

Bottom Line: Characterizing the mode of action of the GAR ESE inside the internal HIV-1 exon 5 we found that this enhancer fulfils a dual splicing regulatory function (i) by synergistically mediating exon recognition through its individual SR protein-binding sites and (ii) by conferring 3' ss selectivity within the 3' ss cluster preceding exon 5.Therefore, a network of cross-exon interactions appears to regulate splicing of the alternative exons 4a and 5.As the GAR ESE-mediated activation of the upstream 3' ss cluster also is essential for the processing of intron-containing vpu/env-mRNAs during intermediate viral gene expression, the GAR enhancer substantially contributes to the regulation of viral replication.

View Article: PubMed Central - PubMed

Affiliation: Institut für Virologie, Universitätsklinikum Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany.

ABSTRACT
The guanosine-adenosine-rich exonic splicing enhancer (GAR ESE) identified in exon 5 of the human immunodeficiency virus type-1 (HIV-1) pre-mRNA activates either an enhancer-dependent 5' splice site (ss) or 3' ss in 1-intron reporter constructs in the presence of the SR proteins SF2/ASF2 and SRp40. Characterizing the mode of action of the GAR ESE inside the internal HIV-1 exon 5 we found that this enhancer fulfils a dual splicing regulatory function (i) by synergistically mediating exon recognition through its individual SR protein-binding sites and (ii) by conferring 3' ss selectivity within the 3' ss cluster preceding exon 5. Both functions depend upon the GAR ESE, U1 snRNP binding at the downstream 5' ss D4 and the E42 sequence located between these elements. Therefore, a network of cross-exon interactions appears to regulate splicing of the alternative exons 4a and 5. As the GAR ESE-mediated activation of the upstream 3' ss cluster also is essential for the processing of intron-containing vpu/env-mRNAs during intermediate viral gene expression, the GAR enhancer substantially contributes to the regulation of viral replication.

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GAR ESE and U1 snRNP binding at D4 are necessary for activation of the 3′ ss cluster in vpu/env-mRNA. (A) RT–PCR analysis of RNA from HeLa-T4+ cells transfected with the 2-inton reporter construct carrying wild-type D4 (D4), a mutant unable to stably bind endogenous U1 snRNA (3U) or a splicing-deficient U1 snRNP-binding site with the additional mutation of the cryptic 5′ ss downstream (GTV 15A). Cells were cotransfected with SVcrev, an expression plasmid for the viral regulatory protein Rev, allowing the export of intron-containing reporter mRNA into the cytoplasm, and with pXGH5. Base pairings between D4 and the splice site mutants 3U and GTV 15A are denoted at the upper panel. The star marks the additional mutation of the cryptic 5′ ss 13-nt downstream of D4 (36). RT–PCR was performed using a Cy5-labelled 5′primer, separated on denaturating gels and detected by (ALF). The RT-PCR products are shown as processed fluorescence curve data of the electrophoretic separation. The alternative spliced mRNA isoforms are depicted above the lanes. The asterisk marks an RNA signal, which was identified as unspecific signal by sequencing. (B) RT–PCR analysis of RNA from HeLa-T4+ cells transiently transfected with the wild-type reporter construct or constructs carrying a single SR protein-binding site (cf. Figure 2A) as indicated in the respective lanes, the Rev-expression plasmid SVcrev and pXGH5. Cy5-labelled RT–PCR products were analysed by ALF.
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Figure 6: GAR ESE and U1 snRNP binding at D4 are necessary for activation of the 3′ ss cluster in vpu/env-mRNA. (A) RT–PCR analysis of RNA from HeLa-T4+ cells transfected with the 2-inton reporter construct carrying wild-type D4 (D4), a mutant unable to stably bind endogenous U1 snRNA (3U) or a splicing-deficient U1 snRNP-binding site with the additional mutation of the cryptic 5′ ss downstream (GTV 15A). Cells were cotransfected with SVcrev, an expression plasmid for the viral regulatory protein Rev, allowing the export of intron-containing reporter mRNA into the cytoplasm, and with pXGH5. Base pairings between D4 and the splice site mutants 3U and GTV 15A are denoted at the upper panel. The star marks the additional mutation of the cryptic 5′ ss 13-nt downstream of D4 (36). RT–PCR was performed using a Cy5-labelled 5′primer, separated on denaturating gels and detected by (ALF). The RT-PCR products are shown as processed fluorescence curve data of the electrophoretic separation. The alternative spliced mRNA isoforms are depicted above the lanes. The asterisk marks an RNA signal, which was identified as unspecific signal by sequencing. (B) RT–PCR analysis of RNA from HeLa-T4+ cells transiently transfected with the wild-type reporter construct or constructs carrying a single SR protein-binding site (cf. Figure 2A) as indicated in the respective lanes, the Rev-expression plasmid SVcrev and pXGH5. Cy5-labelled RT–PCR products were analysed by ALF.

Mentions: To examine the influence of U1 snRNP binding at D4 on activation of the 3′ ss cluster in the late phase, we compared the wild-type 5′ ss D4 with two mutations: 3U abolishing U1 snRNA binding and GTV 15A restitution of U1 snRNP binding but suppressing splicing at D4. The 3U-mutation led to complete loss of vpu/env-mRNA, while the amount of unspliced mRNA remained unchanged, demonstrating that none of the 3′ ss in the cluster upstream of exon 5 had been activated (Figure 6A, cf. 3U versus D4). To confirm that lack of detectability of the vpu/env-mRNA was caused by loss of U1 snRNP binding at D4, we reconstituted U1 snRNP binding by substituting D4 with a splicing inactive U1 snRNP-binding site (GTV) (28). The GTV sequence possesses perfect complementarity to the 5′ end of the U1 snRNA except for position +1, which was mutated from G to C to prevent splicing. After introduction of the GTV sequence, splicing from a cryptic 5′ ss 13-nt downstream of the inactivated 5′ ss (36) was observed (data not shown). To exclude splicing of the downstream intron, we additionally inactivated this cryptic 5′ ss (GTV 15A). Restitution of U1 snRNP binding rescued the expression of vpu/env-mRNA to the same extent as observed with D4 (Figure 6A, cf. GTV 15A versus D4). From this experiment we conclude that U1 snRNP binding at D4 is needed for 3′ ss activation for Rev-dependent mRNAs, although D4 is not spliced itself.Figure 6.


Insights into the selective activation of alternatively used splice acceptors by the human immunodeficiency virus type-1 bidirectional splicing enhancer.

Asang C, Hauber I, Schaal H - Nucleic Acids Res. (2008)

GAR ESE and U1 snRNP binding at D4 are necessary for activation of the 3′ ss cluster in vpu/env-mRNA. (A) RT–PCR analysis of RNA from HeLa-T4+ cells transfected with the 2-inton reporter construct carrying wild-type D4 (D4), a mutant unable to stably bind endogenous U1 snRNA (3U) or a splicing-deficient U1 snRNP-binding site with the additional mutation of the cryptic 5′ ss downstream (GTV 15A). Cells were cotransfected with SVcrev, an expression plasmid for the viral regulatory protein Rev, allowing the export of intron-containing reporter mRNA into the cytoplasm, and with pXGH5. Base pairings between D4 and the splice site mutants 3U and GTV 15A are denoted at the upper panel. The star marks the additional mutation of the cryptic 5′ ss 13-nt downstream of D4 (36). RT–PCR was performed using a Cy5-labelled 5′primer, separated on denaturating gels and detected by (ALF). The RT-PCR products are shown as processed fluorescence curve data of the electrophoretic separation. The alternative spliced mRNA isoforms are depicted above the lanes. The asterisk marks an RNA signal, which was identified as unspecific signal by sequencing. (B) RT–PCR analysis of RNA from HeLa-T4+ cells transiently transfected with the wild-type reporter construct or constructs carrying a single SR protein-binding site (cf. Figure 2A) as indicated in the respective lanes, the Rev-expression plasmid SVcrev and pXGH5. Cy5-labelled RT–PCR products were analysed by ALF.
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Figure 6: GAR ESE and U1 snRNP binding at D4 are necessary for activation of the 3′ ss cluster in vpu/env-mRNA. (A) RT–PCR analysis of RNA from HeLa-T4+ cells transfected with the 2-inton reporter construct carrying wild-type D4 (D4), a mutant unable to stably bind endogenous U1 snRNA (3U) or a splicing-deficient U1 snRNP-binding site with the additional mutation of the cryptic 5′ ss downstream (GTV 15A). Cells were cotransfected with SVcrev, an expression plasmid for the viral regulatory protein Rev, allowing the export of intron-containing reporter mRNA into the cytoplasm, and with pXGH5. Base pairings between D4 and the splice site mutants 3U and GTV 15A are denoted at the upper panel. The star marks the additional mutation of the cryptic 5′ ss 13-nt downstream of D4 (36). RT–PCR was performed using a Cy5-labelled 5′primer, separated on denaturating gels and detected by (ALF). The RT-PCR products are shown as processed fluorescence curve data of the electrophoretic separation. The alternative spliced mRNA isoforms are depicted above the lanes. The asterisk marks an RNA signal, which was identified as unspecific signal by sequencing. (B) RT–PCR analysis of RNA from HeLa-T4+ cells transiently transfected with the wild-type reporter construct or constructs carrying a single SR protein-binding site (cf. Figure 2A) as indicated in the respective lanes, the Rev-expression plasmid SVcrev and pXGH5. Cy5-labelled RT–PCR products were analysed by ALF.
Mentions: To examine the influence of U1 snRNP binding at D4 on activation of the 3′ ss cluster in the late phase, we compared the wild-type 5′ ss D4 with two mutations: 3U abolishing U1 snRNA binding and GTV 15A restitution of U1 snRNP binding but suppressing splicing at D4. The 3U-mutation led to complete loss of vpu/env-mRNA, while the amount of unspliced mRNA remained unchanged, demonstrating that none of the 3′ ss in the cluster upstream of exon 5 had been activated (Figure 6A, cf. 3U versus D4). To confirm that lack of detectability of the vpu/env-mRNA was caused by loss of U1 snRNP binding at D4, we reconstituted U1 snRNP binding by substituting D4 with a splicing inactive U1 snRNP-binding site (GTV) (28). The GTV sequence possesses perfect complementarity to the 5′ end of the U1 snRNA except for position +1, which was mutated from G to C to prevent splicing. After introduction of the GTV sequence, splicing from a cryptic 5′ ss 13-nt downstream of the inactivated 5′ ss (36) was observed (data not shown). To exclude splicing of the downstream intron, we additionally inactivated this cryptic 5′ ss (GTV 15A). Restitution of U1 snRNP binding rescued the expression of vpu/env-mRNA to the same extent as observed with D4 (Figure 6A, cf. GTV 15A versus D4). From this experiment we conclude that U1 snRNP binding at D4 is needed for 3′ ss activation for Rev-dependent mRNAs, although D4 is not spliced itself.Figure 6.

Bottom Line: Characterizing the mode of action of the GAR ESE inside the internal HIV-1 exon 5 we found that this enhancer fulfils a dual splicing regulatory function (i) by synergistically mediating exon recognition through its individual SR protein-binding sites and (ii) by conferring 3' ss selectivity within the 3' ss cluster preceding exon 5.Therefore, a network of cross-exon interactions appears to regulate splicing of the alternative exons 4a and 5.As the GAR ESE-mediated activation of the upstream 3' ss cluster also is essential for the processing of intron-containing vpu/env-mRNAs during intermediate viral gene expression, the GAR enhancer substantially contributes to the regulation of viral replication.

View Article: PubMed Central - PubMed

Affiliation: Institut für Virologie, Universitätsklinikum Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany.

ABSTRACT
The guanosine-adenosine-rich exonic splicing enhancer (GAR ESE) identified in exon 5 of the human immunodeficiency virus type-1 (HIV-1) pre-mRNA activates either an enhancer-dependent 5' splice site (ss) or 3' ss in 1-intron reporter constructs in the presence of the SR proteins SF2/ASF2 and SRp40. Characterizing the mode of action of the GAR ESE inside the internal HIV-1 exon 5 we found that this enhancer fulfils a dual splicing regulatory function (i) by synergistically mediating exon recognition through its individual SR protein-binding sites and (ii) by conferring 3' ss selectivity within the 3' ss cluster preceding exon 5. Both functions depend upon the GAR ESE, U1 snRNP binding at the downstream 5' ss D4 and the E42 sequence located between these elements. Therefore, a network of cross-exon interactions appears to regulate splicing of the alternative exons 4a and 5. As the GAR ESE-mediated activation of the upstream 3' ss cluster also is essential for the processing of intron-containing vpu/env-mRNAs during intermediate viral gene expression, the GAR enhancer substantially contributes to the regulation of viral replication.

Show MeSH
Related in: MedlinePlus