Relative strength of 5’ splice-site strength defines functions of SRSF2 and SRSF6 in alternative splicing of Bcl-x pre-mRNA
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Bcl-x, a member of the Bcl-2 family, plays a key role in apoptosis. Alternative splicing of Bcl-x pre-mRNA through alternative5’ splice-site selection produces an anti-apoptotic mRNAisoform that includes exon 2b and a pro-apoptotic Bcl-x mRNAisoform that excludes exon 2b. Here we used Bcl-x minigeneand identified SRSF2 and SRSF6 as two regulatory factors of 5’splice-site selection of Bcl-x pre-mRNA. We selected bindingclusters closer to 5’ splice-sites from multiple potential bindingsites of SRSF2 and SRSF6 to perform loss of functions analysisthrough site-directed mutagenesis. Our results demonstratedthat these mutations did not abolish regulatory functions ofSRSF2 or SRSF6, indicating that a single binding motif or acluster was not a functional target of these proteins in Bcl-xpre-mRNA splicing. Random deletion mutagenesis did notdisrupt the role of SRSF2 and SRSF6. Importantly, mutagenesisof 5’ splice-site to a conserved or a weaker score demonstratedthat the weaker strength of the target 5’ splice-site or higherstrength of the other 5’ splice-site strength limited the role ofSRSF2 and SRSF6 in 5’ splice-site activation.
Abstract
Bcl-x, a member of the Bcl-2 family, plays a key role in apoptosis. Alternative splicing of Bcl-x pre-mRNA through alternative5’ splice-site selection produces an anti-apoptotic mRNAisoform that includes exon 2b and a pro-apoptotic Bcl-x mRNAisoform that excludes exon 2b. Here we used Bcl-x minigeneand identified SRSF2 and SRSF6 as two regulatory factors of 5’splice-site selection of Bcl-x pre-mRNA. We selected bindingclusters closer to 5’ splice-sites from multiple potential bindingsites of SRSF2 and SRSF6 to perform loss of functions analysisthrough site-directed mutagenesis. Our results demonstratedthat these mutations did not abolish regulatory functions ofSRSF2 or SRSF6, indicating that a single binding motif or acluster was not a functional target of these proteins in Bcl-xpre-mRNA splicing. Random deletion mutagenesis did notdisrupt the role of SRSF2 and SRSF6. Importantly, mutagenesisof 5’ splice-site to a conserved or a weaker score demonstratedthat the weaker strength of the target 5’ splice-site or higherstrength of the other 5’ splice-site strength limited the role ofSRSF2 and SRSF6 in 5’ splice-site activation.
참고문헌 (28)
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