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Utilization of Splicing Elements and Polyadenylation Signal Elements in the Coupling of Polyadenylation and Last-Intron Removal

机译:剪接元件和聚腺苷酸化信号元件在聚腺苷酸化和最后内含子去除耦合中的应用

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摘要

Polyadenylation (PA) is the process by which the 3′ ends of most mammalian mRNAs are formed. In nature, PA is highly coordinated, or coupled, with splicing. In mammalian systems, the most compelling mechanistic model for coupling arises from data supporting exon definition (2, 34, 37). We have examined the roles of individual functional components of splicing and PA signals in the coupling process by using an in vitro splicing and PA reaction with a synthetic pre-mRNA substrate containing an adenovirus splicing cassette and the simian virus 40 late PA signal. The effects of individually mutating splicing elements and PA elements in this substrate were determined. We found that mutation of the polypyrimidine tract and the 3′ splice site significantly reduced PA efficiency and that mutation of the AAUAAA and the downstream elements of the PA signal decreased splicing efficiency, suggesting that these elements are the most significant for the coupling of splicing and PA. Although mutation of the upstream elements (USEs) of the PA signal dramatically decreased PA, splicing was only modestly affected, suggesting that USEs modestly affect coupling. Mutation of the 5′ splice site in the presence of a viable polypyrimidine tract and the 3′ splice site had no effect on PA, suggesting no effect of this element on coupling. However, our data also suggest that a site for U1 snRNP binding (e.g., a 5′ splice site) within the last exon can negatively effect both PA and splicing; hence, a 5′ splice site-like sequence in this position appears to be a modulator of coupling. In addition, we show that the RNA-protein complex formed to define an exon may inhibit processing if the definition of an adjacent exon fails. This finding indicates a mechanism for monitoring the appropriate definition of exons and for allowing only pre-mRNAs with successfully defined exons to be processed.
机译:聚腺苷酸化(PA)是形成大多数哺乳动物mRNA的3'端的过程。从本质上讲,PA是高度协调的或与拼接相结合。在哺乳动物系统中,最引人注目的耦合机理模型来自支持外显子定义的数据(2、34、37)。我们已经通过使用体外剪接和PA与包含腺病毒剪接盒和猿猴病毒40晚期PA信号的合成的pre-mRNA底物的体外剪接和PA反应,研究了剪接和PA信号的各个功能组件在偶联过程中的作用。确定了在该基板中单独改变拼接元素和PA元素的效果。我们发现,聚嘧啶束和3'剪接位点的突变显着降低了PA的效率,而AAUAAA和PA信号的下游元件的突变则降低了剪接效率,这表明这些元件对于剪接和剪接的耦合最为重要。 PA尽管PA信号上游元素(USE)的突变显着降低了PA,但剪接仅受到了适度的影响,这表明USE适度影响了耦合。在有活力的聚嘧啶束的存在下5'剪接位点的突变和3'剪接位点对PA没有影响,表明该元件对偶联没有影响。但是,我们的数据还表明,最后一个外显子中U1 snRNP结合位点(例如5'剪接位点)可能对PA和剪接产生负面影响;因此,在该位置的5'剪接位点样序列似乎是偶联的调节剂。另外,我们显示了形成的用于定义外显子的RNA-蛋白质复合物可能会抑制加工,如果相邻外显子的定义失败。该发现表明了一种机制,用于监测外显子的适当定义并仅允许处理具有成功定义的外显子的前mRNA。

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