Du L, Warren S L
Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
J Cell Biol. 1997 Jan 13;136(1):5-18. doi: 10.1083/jcb.136.1.5.
In the preceding study we found that Sm snRNPs and SerArg (SR) family proteins co-immunoprecipitate with Pol II molecules containing a hyperphosphorylated CTD (Kim et al., 1997). The association between Pol IIo and splicing factors is maintained in the absence of pre-mRNA, and the polymerase need not be transcriptionally engaged (Kim et al., 1997). The latter findings led us to hypothesize that a phosphorylated form of the CTD interacts with pre-mRNA splicing components in vivo. To test this idea, a nested set of CTD-derived proteins was assayed for the ability to alter the nuclear distribution of splicing factors, and to interfere with splicing in vivo. Proteins containing heptapeptides 1-52 (CTD52), 1-32 (CTD32), 1-26 (CTD26), 1-13 (CTD13), 1-6 (CTD6), 1-3 (CTD3), or 1 (CTD1) were expressed in mammalian cells. The CTD-derived proteins become phosphorylated in vivo, and accumulate in the nucleus even though they lack a conventional nuclear localization signal. CTD52 induces a selective reorganization of splicing factors from discrete nuclear domains to the diffuse nucleoplasm, and significantly, it blocks the accumulation of spliced, but not unspliced, human beta-globin transcripts. The extent of splicing factor disruption, and the degree of inhibition of splicing, are proportional to the number of heptapeptides added to the protein. The above results indicate a functional interaction between Pol II's CTD and pre-mRNA splicing.
在之前的研究中，我们发现Sm核小核糖核蛋白颗粒（snRNPs）和丝氨酸精氨酸（SerArg，SR）家族蛋白与含有高度磷酸化羧基末端结构域（CTD）的RNA聚合酶II（Pol II）分子发生共免疫沉淀（Kim等人，1997年）。在没有前体mRNA的情况下，Pol IIo与剪接因子之间的关联依然存在，并且这种聚合酶无需参与转录（Kim等人，1997年）。后面这些发现让我们推测，CTD的磷酸化形式在体内与前体mRNA剪接成分相互作用。为了验证这一想法，我们检测了一组嵌套的源自CTD的蛋白质，看它们是否能够改变剪接因子在细胞核内的分布，以及在体内干扰剪接过程。含有七肽1 - 52（CTD52）、1 - 32（CTD32）、1 - 26（CTD26）、1 - 13（CTD13）、1 - 6（CTD6）、1 - 3（CTD3）或1（CTD1）的蛋白质在哺乳动物细胞中表达。源自CTD的蛋白质在体内发生磷酸化，并且即使它们缺乏传统的核定位信号，也会在细胞核中积累。CTD52诱导剪接因子从离散的核结构域选择性地重新分布到弥散的核质中，并且重要的是，它会阻断已剪接的人β-珠蛋白转录本的积累，但不会阻断未剪接的转录本。剪接因子的破坏程度以及剪接抑制程度与添加到蛋白质中的七肽数量成正比。上述结果表明Pol II的CTD与前体mRNA剪接之间存在功能上的相互作用。
