Spliceosome specifically associates with CTD phospho-serine 5 isoform of RNA polymerase II to promote co-transcriptional splicing

Co-transcriptional splicing of introns is a defining feature of eukaryotic gene expression. We show that the mammalian spliceosome specifically associates with the S5P CTD isoform of RNA polymerase II (Pol II) as it elongates across spliced exons of protein coding genes, both in human Hela and murine lymphoid cell lines. Immuno-precipitation of MNase digested chromatin with phospho CTD specific antibodies reveals that components of the active spliceosome (both snRNA and proteins) form a specific complex with S5P CTD Pol II. Furthermore a dominant splicing intermediate formed by cleavage at intron 5’ss results in the tethering of upstream exons to this complex at all spliced exons. These are invariably connected to upstream spliced constitutive and less frequently to alternative exons. Finally S5P CTD Pol II accumulates over spliced exons but not adjacent introns. We propose that mammalian splicing employs a rapid, co-transcriptional splicing mechanism based on CTD phosphorylation transitions.

内含子的共转录剪接（co-transcriptional splicing）是真核基因表达的标志性特征。本研究证实，在人类HeLa细胞与小鼠淋巴样细胞系中，哺乳动物剪接体（spliceosome）会在RNA聚合酶II（RNA polymerase II，Pol II）沿蛋白质编码基因的剪接型外显子延伸时，特异性结合其S5磷酸化的羧基末端结构域（C-terminal domain，CTD）亚型。使用靶向磷酸化CTD的特异性抗体对经微球菌核酸酶（micrococcal nuclease，MNase）消化的染色质进行免疫沉淀（immunoprecipitation）实验表明，活性剪接体的组分（包括小核RNA（small nuclear RNA，snRNA）与蛋白质）可与S5P-CTD修饰的Pol II形成特异性复合物。进一步研究发现，在内含子5'剪接位点（splice site，ss）处切割形成的主要剪接中间产物，会将上游外显子锚定至所有剪接型外显子处的该复合物中。这类锚定事件通常与上游剪接型组成型外显子相连，仅极少情况下会关联可变外显子。最终，S5P-CTD修饰的Pol II会在剪接型外显子区域富集，而非相邻的内含子区域。据此我们提出，哺乳动物剪接过程依赖基于CTD磷酸化转换的快速共转录剪接机制。