Mutational analysis of Trypanosoma brucei editosome proteins KREPB4 and KREPB5 reveals domains critical for function
Mutational analysis of Trypanosoma brucei editosome proteins KREPB4 and KREPB5 reveals domains critical for function
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DOI：
10.1261/rna.035048.112
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发表时间：
2012-10-01
期刊：
影响因子：
4.5
通讯作者：
Stuart, Kenneth
中科院分区：
文献类型：
--
作者：
Carnes, Jason;Schnaufer, Achim;Stuart, Kenneth
动质体线粒体的转录组经历插入和缺失尿苷残基（U）的广泛RNA编辑以产生成熟mRNA。编辑体是一种多蛋白复合物，其提供RNA编辑所需的核酸内切酶、TUTase、核酸外切酶和连接酶活性。编辑体的KREPB 4和KREPB 5蛋白对于编辑体完整性和寄生虫生存力是必需的，并且含有对应于锌指、RNA酶III和PUF结构域的半保守基序，但是迄今为止没有报道这些结构域的功能分析。我们在这里展示了KREPB 4和KREPB 5的各种点突变识别了必需的结构域，并表明这些蛋白质本身不进行RNA酶III催化。KREPB 4而不是KREPB 5的锌指是编辑体完整性和寄生虫生存力所必需的，并且KREPB 5中RNase III特征基序的突变阻止整合到编辑体中，这是致命的。分离的TAP标记的KREPB 4和KREPB 5复合物优先与缺失亚复合物的组分相关联，为编辑体结构提供了额外的见解。来自几种动质体物种的编辑体RNA酶III序列的新比对表明KREPB 4和KREPB 5缺乏催化活性，并揭示了PUF基序存在于编辑核酸内切酶KREN1，KREN2和KREN3中。这里呈现的数据与KREPB 4和KREPB 5与催化活性编辑核酸内切酶形成分子间异二聚体的假设一致，这在已知的RNase III蛋白中是前所未有的。
The transcriptome of kinetoplastid mitochondria undergoes extensive RNA editing that inserts and deletes uridine residues (U's) to produce mature mRNAs. The editosome is a multiprotein complex that provides endonuclease, TUTase, exonuclease, and ligase activities required for RNA editing. The editosome's KREPB4 and KREPB5 proteins are essential for editosome integrity and parasite viability and contain semi-conserved motifs corresponding to zinc finger, RNase III, and PUF domains, but to date no functional analysis of these domains has been reported. We show here that various point mutations to KREPB4 and KREPB5 identify essential domains, and suggest that these proteins do not themselves perform RNase III catalysis. The zinc finger of KREPB4 but not KREPB5 is essential for editosome integrity and parasite viability, and mutation of the RNase III signature motif in KREPB5 prevents integration into editosomes, which is lethal. Isolated TAP-tagged KREPB4 and KREPB5 complexes preferentially associate with components of the deletion subcomplex, providing additional insights into editosome architecture. A new alignment of editosome RNase III sequences from several kinetoplastid species implies that KREPB4 and KREPB5 lack catalytic activity and reveals that the PUF motif is present in the editing endonucleases KREN1, KREN2, and KREN3. The data presented here are consistent with the hypothesis that KREPB4 and KREPB5 form intermolecular heterodimers with the catalytically active editing endonucleases, which is unprecedented among known RNase III proteins.