An evolutionarily conserved role for SRm160 in 3′-end processing that functions independently of exon junction complex formation
An evolutionarily conserved role for SRm160 in 3′-end processing that functions independently of exon junction complex formation
复制标题
DOI：
10.1074/jbc.m306856200
复制
发表时间：
2003-11-07
影响因子：
4.8
通讯作者：
Blencowe, BJ
中科院分区：
文献类型：
--
作者：
McCracken, S;Longman, D;Blencowe, BJ
SRm160（160 kDa 的 SR 相关核基质蛋白）充当剪接共激活剂和 3' 端裂解刺激因子。它也是剪接依赖性外显子连接复合物 (EJC) 的组成部分，该复合物参与前 mRNA 剪接与 mRNA 周转和 mRNA 输出的耦合。我们研究了 SRm160 与 EJC 的关联对于有效的 3' 末端切割是否重要。 EJC 组件 RNPS1、REF、UAP56 和 Y14 与 SRm160 交互。然而，当这些因子与转录物结合时，只有 SRm160 和 RNPS1 刺激 3' 端切割。尽管 SRm160 在存在或不存在活性内含子的情况下刺激切割达到类似程度，但束缚的 RNPS1 对 3' 末端切割的刺激依赖于活性内含子。体外在切割和聚腺苷酸化信号附近组装 EJC 不会显着影响切割效率。这些结果表明，SRm160 刺激裂解，与其与 EJC 成分的关联无关，并且 RNPS1 的裂解刺激活性可能是其刺激剪接能力的间接结果。利用秀丽隐杆线虫中的 RNA 干扰 (RNAi)，我们确定了 SRm160 和裂解机制之间的相互作用在整个生物体环境中是否重要。 SRm160 和裂解因子 CstF-50（裂解刺激因子 50-kDa 亚基）的同时 RNAi 导致晚期胚胎发育停滞。相反，CstF-50 的 RNAi 与 RNPS1 或 REF 组合并没有产生明显的表型。我们的综合结果为 SRm160 和独立于 EJC 形成发挥作用的 3' 端裂解机制之间进化上保守的相互作用提供了证据。
SRm160 (the SR-related nuclear matrix protein of 160 kDa) functions as a splicing coactivator and 3'-end cleavage-stimulatory factor. It is also a component of the splicing-dependent exon-junction complex (EJC), which has been implicated in coupling of pre-mRNA splicing with mRNA turnover and mRNA export. We have investigated whether the association of SRm160 with the EJC is important for efficient 3'-end cleavage. The EJC components RNPS1, REF, UAP56, and Y14 interact with SRm160. However, when these factors were tethered to transcripts, only SRm160 and RNPS1 stimulated 3'-end cleavage. Whereas SRm160 stimulated cleavage to a similar extent in the presence or absence of an active intron, stimulation of 3'-end cleavage by tethered RNPS1 is dependent on an active intron. Assembly of an EJC adjacent to the cleavage and polyadenylation signal in vitro did not significantly affect cleavage efficiency. These results suggest that SRm160 stimulates cleavage independently of its association with EJC components and that the cleavage-stimulatory activity of RNPS1 may be an indirect consequence of its ability to stimulate splicing. Using RNA interference (RNAi) in Caenorhabditis elegans, we determined whether interactions between SRm160 and the cleavage machinery are important in a whole organism context. Simultaneous RNAi of SRm160 and the cleavage factor CstF-50 ( Cleavage stimulation factor 50-kDa subunit) resulted in late embryonic developmental arrest. In contrast, RNAi of CstF-50 in combination with RNPS1 or REFs did not result in an apparent phenotype. Our combined results provide evidence for an evolutionarily conserved interaction between SRm160 and the 3'-end cleavage machinery that functions independently of EJC formation.