Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particle.
Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particle.
复制标题
通过 U1 小核核糖核蛋白颗粒的两个位点结合进行外显子对的组合剪接。
DOI：
10.1128/mcb.11.12.5919-5928.1991
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发表时间：
1991
影响因子：
5.3
通讯作者：
Burch,R
中科院分区：
文献类型：
--
作者：
Grabowski,PJ;Nasim,FU;Kuo,HC;Burch,R
为了了解在含有替代外显子的复杂前mRNAs中如何选择外显子伙伴，对U1小核核糖核蛋白颗粒(U1 SnRNP)结合的双位点模型进行了测试。在该模型中，提出了两个U1SnRNPs通过与下游外显子的3‘边界和待剪接内含子的5’边界进行碱基配对来定义一个剪接功能单位。三外显子底物含有速激肽前基因的外显子4(E4)区域。相邻外显子5‘端剪接位点的联合突变表明，U1 SnRNP在下游位点的结合减弱，而U1 SnRNP在上游位点的结合增强，导致未能挽救突变之间内含子的剪接。这些结果表明，对于这些底物，将第二个U1 SnRNP结合到下游5‘剪接位点的要求是严格的，而不是在任一位置都可以提供特定阈值水平的U1 SnRNP的替代模型。第三个外显子E5的5‘剪接点的单位点突变削弱了与Ul RNA的碱基互补性，进一步支持了双位点模型。这些突变阻止了E5分支点的形成，令人惊讶的是，产生了新的分支点，这些分支点主要由它们与位于Pre-mRNA 3‘末端的神秘的5’剪接位点的接近而指定。实验证明了U1 SnRNP下游结合对3‘剪接位点活性的真实刺激，并提出了一种可能的机制，通过该机制可以实现选择性剪接的前mRNAs的外显子选择的组合模式。
A two-site model for the binding of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was tested in order to understand how exon partners are selected in complex pre-mRNAs containing alternative exons. In this model, it is proposed that two U1 snRNPs define a functional unit of splicing by base pairing to the 3' boundary of the downstream exon as well as the 5' boundary of the intron to be spliced. Three-exon substrates contained the alternatively spliced exon 4 (E4) region of the preprotachykinin gene. Combined 5' splice site mutations at neighboring exons demonstrate that weakened binding of U1 snRNP at the downstream site and improved U1 snRNP binding at the upstream site result in the failure to rescue splicing of the intron between the mutations. These results indicate the stringency of the requirement for binding a second U1 snRNP to the downstream 5' splice site for these substrates as opposed to an alternative model in which a certain threshold level of U1 snRNP can be provided at either site. Further support for the two-site model is provided by single-site mutations in the 5' splice site of the third exon, E5, that weaken base complementarity to Ul RNA. These mutations block E5 branchpoint formation and, surprisingly, generate novel branchpoints that are specified chiefly by their proximity to a cryptic 5' splice site located at the 3' terminus of the pre-mRNA. The experiments shown here demonstrate a true stimulation of 3' splice site activity by the downstream binding of U1 snRNP and suggest a possible mechanism by which combinatorial patterns of exon selection are achieved for alternatively spliced pre-mRNAs.