Prp45 Affects Prp22 Partition in Spliceosomal Complexes and Splicing Efficiency of Non-Consensus Substrates
Prp45 Affects Prp22 Partition in Spliceosomal Complexes and Splicing Efficiency of Non-Consensus Substrates
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DOI：
10.1002/jcb.21989
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发表时间：
2009-01-01
影响因子：
4
通讯作者：
Puta, Frantisek
中科院分区：
文献类型：
--
作者：
Gahura, Ondrej;Abrhamova, Katerina;Puta, Frantisek
人类转录辅助调节因子SNW 1/SKIP参与转录延伸和选择性剪接的调节。Prp 45是酵母中的SNW/SKIP直系同源物，被认为是前mRNA加工所必需的。在这里，我们描述了pip 45（1-169），一个PRP 45的温度敏感等位基因，在允许的温度下，它会导致细胞分裂缺陷和对微管抑制剂的超敏反应。使用合成致死筛选，我们发现prp 45（1-169）与NTC成员SYF 1、CLF 1/SYF 3、NTC 20和CEF 1的等位基因以及第二步剪接因子SLU 7、PRP 17、PRP 18和PRP 22的等位基因发生遗传相互作用。从prp 45（1-169）细胞中纯化的Cwc 2相关剪接体复合物显示Prp 22的化学计量减少，表明其与剪接体的相互作用紊乱。在prp 45（1-169）细胞中的体内剪接测定揭示，与野生型细胞相比，分支点突变体积累更多的前mRNA，而5'和3'剪接位点突变体显示出拉里司松中间体水平升高。典型内含子的剪接不受阻碍。值得注意的是，prp 45（1-169）细胞中Prp 45（119-379）的表达恢复了Cwc 2-下拉中的Prp 22分配，并挽救了prp 45（1-169）菌株的温度敏感性和剪接表型。我们的数据表明，Prp 45有助于，部分通过其与第二步校对解旋酶Prp 22的相互作用，剪接效率的基板不符合共识。J.细胞。106：139-151，2009. (C)2008 Wiley-Liss，Inc.
Human transcription co-regulator SNW1/SKIP is implicated in the regulation of both transcription elongation and alternative splicing. Prp45, the SNW/SKIP ortholog in yeast, is assumed to be essential for pre-mRNA processing. Here, we characterize pip45(1-169), a temperature sensitive allele of PRP45, which at permissive temperature elicits cell division defects and hypersensitivity to microtubule inhibitors. Using a synthetic lethality screen, we found that prp45(1-169) genetically interacts with alleles of NTC members SYF1, CLF1/SYF3, NTC20, and CEF1, and 2nd step splicing factors SLU7, PRP17, PRP18, and PRP22. Cwc2-associated spliceosomal complexes purified from prp45(1-169) cells showed decreased stoichiometry of Prp22, suggesting its deranged interaction with the spliceosome. In vivo splicing assays in prp45(1-169) cells revealed that branch point mutants accumulated more pre-mRNA whereas 5' and 3' splice site mutants showed elevated levels of lariatexon intermediate as compared to wild-type cells. Splicing of canonical intron was unimpeded. Notably, the expression of Prp45(119-379) in prp45(1-169) cells restored Prp22 partition in the Cwc2-pulldowns and rescued temperature sensitivity and splicing phenotype of prp45(1-169) strain. Our data suggest that Prp45 contributes, in part through its interaction with the 2nd step-proofreading helicase Prp22, to splicing efficiency of substrates non-conforming to the consensus. J. Cell. Biochem. 106: 139-151, 2009. (C) 2008 Wiley-Liss, Inc.