Alvi R K, Lund M, Okeefe R T
School of Biological Sciences, University of Manchester, United Kingdom.
RNA. 2001 Jul;7(7):1013-23. doi: 10.1017/s135583820101041x.
Pre-messenger RNA splicing is a two-step process by which introns are removed and exons joined together. In yeast, the U5 snRNA loop 1 interacts with the 5' exon before the first step of splicing and with the 5' and 3' exons before the second step. In vitro studies revealed that yeast U5 loop 1 is not required for the first step of splicing but is essential for holding the 5' and 3' exons for ligation during the second step. It is critical, therefore, that loop 1 contacts the 5' exon before the first step of splicing to hold this exon following cleavage from the pre-mRNA. At present it is not known how U5 loop 1 is positioned on the 5' exon prior to the first step of splicing. To address this question, we have used site-specific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of U5 loop 1 with the pre-mRNA prior to the first step of splicing. We have found that the highly conserved uridines in loop 1 make ATP-dependent contacts with an approximately 8-nt region at the 5' splice site that includes the invariant GU. These interactions are dependent on functional U2 and U6 snRNAs. Our results support a model where U5 snRNA loop 1 interacts with the 5' exon in two steps during its targeting to the 5' splice site.
信使前体RNA剪接是一个两步过程，通过该过程内含子被去除，外显子连接在一起。在酵母中，U5小核仁RNA（snRNA）环1在剪接第一步之前与5'外显子相互作用，在第二步之前与5'和3'外显子相互作用。体外研究表明，酵母U5环1对于剪接的第一步不是必需的，但对于在第二步中保持5'和3'外显子进行连接至关重要。因此，至关重要的是，环1在剪接第一步之前与5'外显子接触，以便在从前体mRNA切割后保持该外显子。目前尚不清楚U5环1在剪接第一步之前如何定位在5'外显子上。为了解决这个问题，我们在酵母剪接体中使用了位点特异性光活化交联，以研究U5环1在剪接第一步之前与前体mRNA的相互作用。我们发现，环1中高度保守的尿苷与5'剪接位点处约8个核苷酸的区域进行ATP依赖性接触，该区域包括不变的GU。这些相互作用依赖于功能性U2和U6 snRNA。我们的结果支持一个模型，其中U5 snRNA环1在靶向5'剪接位点的过程中分两步与5'外显子相互作用。
