The P-Loop Domain of Yeast Clp1 Mediates Interactions Between CF IA and CPF Factors in Pre-mRNA 3′ End Formation
The P-Loop Domain of Yeast Clp1 Mediates Interactions Between CF IA and CPF Factors in Pre-mRNA 3′ End Formation
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DOI：
10.1371/journal.pone.0029139
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发表时间：
2011-12-22
期刊：
影响因子：
3.7
通讯作者：
Dichtl, Bernhard
中科院分区：
文献类型：
--
作者：
Holbein, Sandra;Scola, Simonetta;Dichtl, Bernhard
裂解因子IA（CFIA）、裂解和多聚腺苷酸化因子（CPF）构成酵母中前体mRNA 3'末端形成所需的主要蛋白质复合物。Clp 1蛋白与CF IA中的Pcf 11、Rna 15和Rna 14相关，但其功能作用尚不清楚。Clp 1携带一个进化上保守的P环基序，该基序先前被证明可以结合ATP。有趣的是，人类和古细菌Clp 1同源物，而不是酵母蛋白，携带5' RNA激酶活性。我们发现，在酵母中的Clp 1的消耗促进缺陷的3'端形成和RNA聚合酶II终止，然而，细胞表达的Clp 1与突变的P-环显示只有轻微的缺陷，基因表达。类似地，干扰ATP结合的纯化和重建的突变CF IA因子在偶联的体外转录/3'末端加工反应中补充CF IA耗尽的提取物。我们发现，Clp 1需要组装重组CF IA和某些P-环突变体未能与CF IA亚基Pcf 11相互作用。相比之下，Clp 1突变增强了与CPF组分3'内切核酸酶Ysh 1的结合。我们的研究结果支持Clp 1 P-环基序的结构作用。Clp 1与ATP的结合可能有助于CF IA的形成和3'末端形成过程中的交叉因子相互作用。
Cleavage factor IA (CF IA), cleavage and polyadenylation factor (CPF), constitute major protein complexes required for pre-mRNA 3' end formation in yeast. The Clp1 protein associates with Pcf11, Rna15 and Rna14 in CF IA but its functional role remained unclear. Clp1 carries an evolutionarily conserved P-loop motif that was previously shown to bind ATP. Interestingly, human and archaean Clp1 homologues, but not the yeast protein, carry 5' RNA kinase activity. We show that depletion of Clp1 in yeast promoted defective 3' end formation and RNA polymerase II termination; however, cells expressing Clp1 with mutant P-loops displayed only minor defects in gene expression. Similarly, purified and reconstituted mutant CF IA factors that interfered with ATP binding complemented CF IA depleted extracts in coupled in vitro transcription/3' end processing reactions. We found that Clp1 was required to assemble recombinant CF IA and that certain P-loop mutants failed to interact with the CF IA subunit Pcf11. In contrast, mutations in Clp1 enhanced binding to the 3' endonuclease Ysh1 that is a component of CPF. Our results support a structural role for the Clp1 P-loop motif. ATP binding by Clp1 likely contributes to CF IA formation and cross-factor interactions during the dynamic process of 3' end formation.