Functional coupling of cleavage and polyadenylation with transcription of mRNA
Functional coupling of cleavage and polyadenylation with transcription of mRNA
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DOI：
10.1074/jbc.m505532200
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发表时间：
2005-09-16
影响因子：
4.8
通讯作者：
Price, DH
中科院分区：
文献类型：
--
作者：
Adamson, TE;Shutt, DC;Price, DH
几乎所有真核生物mRNAs的3‘端都由切割和多聚腺苷作用决定，它们被认为是在转录过程中发生的。我们描述了一种利用固定化模板的人体体外系统，在该系统中，RNA聚合酶II延伸复合体中的转录本被有效地切割和多腺化。由于游离RNA的切割速度要慢得多，我们得出结论，切割与转录是功能耦合的。抑制正转录延伸因子b(P-TEFb)对切割只有轻微的负面影响，只要转录本足够长，包含多聚腺苷酸化信号。相反，去除RNA聚合酶II大亚基的羧基末端结构域对切割有戏剧性的负面影响。出乎意料的是，切割后的转录本的5‘端部分仍然与模板在一个功能性的、具有多聚腺苷酸化能力的复合体中相关联。有效的切割需要人的封端酶对5‘端进行封端，但在COOH末端无结构域聚合酶延伸复合体中转录物的切割减少并不是因为缺乏封端。
Cleavage and polyadenylation define the 3' ends of almost all eukaryotic mRNAs and are thought to occur during transcription. We describe a human in vitro system utilizing an immobilized template, in which transcripts in RNA polymerase II elongation complexes are efficiently cleaved and polyadenylated. Because the cleavage rate of free RNA is much slower, we conclude that cleavage is functionally coupled to transcription. Inhibition of positive transcription elongation factor b(P-TEFb) had only a modest negative effect on cleavage, as long as transcripts were long enough to contain the polyadenylation signal. In contrast, removal of the carboxyl-terminal domain of the large subunit of RNA polymerase II had a dramatic negative effect on cleavage. Unexpectedly, the 5' portion of transcript after cleavage remained associated with the template in a functional, polyadenylation-competent complex. Efficient cleavage required 5' capping by the human capping enzyme, but the reduction of cleavage seen of transcripts in COOH-terminal domain-less polymerase elongation complexes, was not because of lack of capping.