A complex containing CstF-64 and the SL2 snRNP connects mRNA 3′ end formation and trans-splicing in C-elegans operons
A complex containing CstF-64 and the SL2 snRNP connects mRNA 3′ end formation and trans-splicing in C-elegans operons
复制标题
DOI：
10.1101/gad.920501
复制
发表时间：
2001-10-01
影响因子：
10.5
通讯作者：
Blumenthal, T
中科院分区：
文献类型：
--
作者：
Evans, D;Perez, I;Blumenthal, T
秀丽隐杆线虫的多顺反子前体mRNA通过上游mRNA的3'末端形成和下游mRNA的SL 2特异性反式剪接来加工。这些过程通常发生在约100个核苷酸的区域内，并且是机械偶联的。在本文中，我们报道了一个复杂的C。含有3 ′末端形成蛋白CstF-64和SL 2 snRNP的线虫提取物。这种与α CstF-64抗体免疫沉淀的复合物含有SL 2 RNA，但不含有SL 1 RNA或其他U snRNA。使用突变分析，我们已经能够解偶联SL 2 snRNP功能和身份。茎/环III中具有突变的SL 2 RNA在体内具有反式剪接供体的功能，但未能剪接到SL 2接受反式剪接位点，这表明它已经失去了作为SL 2 snRNP的身份。重要的是，茎/环III突变阻止SL 2 RNA与CstF-64的缔合。相比之下，使SL 2 snRNP失活的茎II中的突变仍然允许与CstF-64形成复合物。因此，SL 2 RNA茎/环III是SL 2身份和含有CstF-64的复合物的形成所需的，但不是反式剪接所需的。这些结果为C.线虫操纵子
Polycistronic pre-mRNAs from Caenorhabditis elegans are processed by 3' end formation of the upstream mRNA and SL2-specffic trans-splicing of the downstream mRNA. These processes usually occur within an -100-nucleotide region and are mechanistically coupled. In this paper, we report a complex in C. elegans extracts containing the 3' end formation protein CstF-64 and the SL2 snRNP. This complex, immunoprecipitated,with alpha CstF-64 antibody, contains SL2 RNA, but not SL1 RNA or other U snRNAs. Using mutational analysis we have been able to uncouple SL2 snRNP function and identity. SL2 RNA with a mutation in stem/loop III is functional in vivo as a trans-splice donor, but fails to splice to SL2-accepting trans-splice sites, suggesting that it has lost its identity as an SL2 snRNP. Importantly, stem/loop III mutations prevent association of SL2 RNA with CstF-64. In contrast, a mutation in stem II that inactivates the SL2 snRNP still permits complex formation with CstF-64. Therefore, SL2 RNA stem/loop III is required for both SL2 identity and formation of a complex containing CstF-64, but not for trans-splicing. These results provide a molecular framework for the coupling of 3' end formation and trans-splicing in the processing of polycistronic pre-mRNAs from C. elegans operons.