Participation of the C-terminal domain of RNA polymerase II in exon definition during pre-mRNA splicing.
Participation of the C-terminal domain of RNA polymerase II in exon definition during pre-mRNA splicing.
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RNA 聚合酶 II 的 C 端结构域在前 mRNA 剪接过程中参与外显子定义。
DOI：
10.1128/mcb.20.21.8290-8301.2000
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发表时间：
2000
影响因子：
5.3
通讯作者：
Berget,SM
中科院分区：
文献类型：
--
作者：
Zeng,C;Berget,SM
转录和前体mRNA加工之间的相互作用，通过结合聚合酶II（Pol II）的因素，涉及加帽，剪接，和聚腺苷酸化最近已被证明。C-末端结构域（CTD），Pol II的最大亚基的高度磷酸化的重复序列，已经涉及这种相互作用，因为该结构域的缺失影响下游RNA加工事件，并且因为它是许多加工因子的结合位点。在这里，我们表明，重组CTD，不含Pol II的其他组分，激活体外剪接和核提取物中剪接体的组装，如果且仅如果，通过外显子定义识别测定的前体RNA，即，如果底物含有同时具有3′和5′剪接位点的完整外显子。此外，消耗完整的Pol II使这组前体RNA的剪接失活，并且添加重组CTD恢复活性。添加的重组CTD在提取物中快速地被高磷酸化和低磷酸化，与前体RNA结合，并刺激U1 snRNP而不是ASF/SF 2与底物RNA的结合。这些观察结果表明，CTD和剪接因子之间的相互作用的模式是完整地绑定到外显子的定义和机制，从而远端外显子可以被识别，并在剪接体的组装过程中进入并列。
Interaction between transcription and pre-mRNA processing via binding of polymerase II (Pol II) to factors involved in capping, splicing, and polyadenylation has recently been demonstrated. The C-terminal domain (CTD), a highly phosphorylated repeat sequence of the largest subunit of Pol II, has been implicated in this interaction because deletion of this domain affects downstream RNA processing events and because it is the binding site for numerous processing factors. Here we show that recombinant CTD, free of other components of Pol II, activated in vitro splicing and assembly of the spliceosome in nuclear extracts if, and only if, the assayed precursor RNA was recognized via exon definition, i.e., if the substrates contained complete exons with both 3′ and 5′ splice sites. Furthermore, depletion of intact Pol II inactivated splicing of this set of precursor RNAs and addition of recombinant CTD restored activity. The added recombinant CTD was quickly hyper- and hypophosphorylated in extract, became associated with the precursor RNA, and stimulated the association of U1 snRNPs but not ASF/SF2 with substrate RNA. These observations suggest that the mode of interaction between the CTD and splicing factors is integrally tied to exon definition and the mechanism whereby distal exons can be recognized and brought into juxtaposition during assembly of the spliceosome.