School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 61005, Korea.
Department of Surgery, Seoul National University College of Medicine, Seoul 03080, Korea.
BMB Rep. 2021 Mar;54(3):176-181. doi: 10.5483/BMBRep.2021.54.3.170.
Bcl-x, a member of the Bcl-2 family, plays a key role in apoptosis. Alternative splicing of Bcl-x pre-mRNA through alternative 5' splice-site selection produces an anti-apoptotic mRNA isoform that includes exon 2b and a pro-apoptotic Bcl-x mRNA isoform that excludes exon 2b. Here we used Bcl-x minigene and identified SRSF2 and SRSF6 as two regulatory factors of 5' splice-site selection of Bcl-x pre-mRNA. We selected binding clusters closer to 5' splice-sites from multiple potential binding sites of SRSF2 and SRSF6 to perform loss of functions analysis through site-directed mutagenesis. Our results demonstrated that these mutations did not abolish regulatory functions of SRSF2 or SRSF6, indicating that a single binding motif or a cluster was not a functional target of these proteins in Bcl-x pre-mRNA splicing. Random deletion mutagenesis did not disrupt the role of SRSF2 and SRSF6. Importantly, mutagenesis of 5' splice-site to a conserved or a weaker score demonstrated that the weaker strength of the target 5' splice-site or higher strength of the other 5' splice-site strength limited the role of SRSF2 and SRSF6 in 5' splice-site activation. [BMB Reports 2021; 54(3): 176-181].
Bcl-x 是 Bcl-2 家族的一员，在细胞凋亡中起着关键作用。Bcl-x 前体 mRNA 通过选择性 5' 剪接位点的选择进行选择性剪接，产生一种抗凋亡的 mRNA 异构体，该异构体包含外显子 2b 和一种促凋亡的 Bcl-x mRNA 异构体，该异构体不包含外显子 2b。在这里，我们使用了 Bcl-x 基因，并鉴定出 SRSF2 和 SRSF6 是 Bcl-x 前体 mRNA 5' 剪接位点选择的两个调节因子。我们从 SRSF2 和 SRSF6 的多个潜在结合位点中选择靠近 5' 剪接位点的结合簇，通过定点诱变进行功能丧失分析。结果表明，这些突变并没有消除 SRSF2 或 SRSF6 的调节功能，这表明单个结合基序或簇不是这些蛋白质在 Bcl-x 前体 mRNA 剪接中的功能靶标。随机缺失突变没有破坏 SRSF2 和 SRSF6 的作用。重要的是，5' 剪接位点的诱变至保守或较弱的评分表明，较弱的靶 5' 剪接位点强度或其他 5' 剪接位点强度较高会限制 SRSF2 和 SRSF6 在 5' 剪接位点激活中的作用。[BMB 报告 2021;54(3):176-181]。
