Human RNA 5′-kinase (hClp1) can function as a tRNA splicing enzyme in vivo
Human RNA 5′-kinase (hClp1) can function as a tRNA splicing enzyme in vivo
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DOI：
10.1261/rna.1142908
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发表时间：
2008-09-01
期刊：
影响因子：
4.5
通讯作者：
Schwer, Beate
中科院分区：
文献类型：
--
作者：
Ramirez, Alejandro;Shuman, Stewart;Schwer, Beate
酵母和人类Clp1蛋白是mRNA 3'-裂解-聚腺苷酸化机制的同源组分。最近的研究强调了人类Clp1 （hClp1）与tRNA剪接内切酶和hClp1内在的rna特异性5'-OH多核苷酸激酶活性的关联，这促使人们猜测Clp1可能在动物细胞中tRNA剪接中起催化作用。在这里，我们发现hClp1在出芽酵母中的表达可以补充酵母或植物tRNA连接酶必需的5'-OH RNA激酶模块的条件和致死突变。酵母中hClp1的tRNA剪接活性被激酶活性位点的突变所消除。相比之下，酵母Clp1 （yClp1）的过表达不能挽救激酶缺陷的tRNA连接酶突变体，并且与hClp1不同，纯化的重组yClp1蛋白在体外没有可检测到的RNA激酶活性。yClp1 atp结合位点的突变不影响酵母的生存能力。这些发现，以及hClp1不能补充酵母clp1 δ菌株生长的事实表明，酵母和人类clp1蛋白尽管结构相似，但在功能上不是同源的。虽然hClp1可以在体内完成酵母型tRNA剪接途径的5'端愈合步骤，但考虑到酵母型tRNA修复酶的其他哺乳动物对应物在体内不是必需的，因此尚不确定其激酶活性是否对人类细胞中的tRNA剪接是必需的。
Yeast and human Clp1 proteins are homologous components of the mRNA 3'-cleavage-polyadenylation machinery. Recent studies highlighting an association of human Clp1 (hClp1) with tRNA splicing endonuclease and an intrinsic RNA-specific 5'-OH polynucleotide kinase activity of hClp1 have prompted speculation that Clp1 might play a catalytic role in tRNA splicing in animal cells. Here, we show that expression of hClp1 in budding yeast can complement conditional and lethal mutations in the essential 5'-OH RNA kinase module of yeast or plant tRNA ligases. The tRNA splicing activity of hClp1 in yeast is abolished by mutations in the kinase active site. In contrast, overexpression of yeast Clp1 (yClp1) cannot rescue kinase-defective tRNA ligase mutants, and, unlike hClp1, the purified recombinant yClp1 protein has no detectable RNA kinase activity in vitro. Mutations of the yClp1 ATP-binding site do not affect yeast viability. These findings, and the fact that hClp1 cannot complement growth of a yeast clp1 Delta strain, indicate that yeast and human Clp1 proteins are not functional orthologs, despite their structural similarity. Although hClp1 can perform the 5'-end-healing step of a yeast-type tRNA splicing pathway in vivo, it is uncertain whether its kinase activity is necessary for tRNA splicing in human cells, given that other mammalian counterparts of yeast-type tRNA repair enzymes are nonessential in vivo.