Aphasizheva Inna, Ringpis Gene-Errol, Weng James, Gershon Paul D, Lathrop Richard H, Aphasizhev Ruslan
Department of Microbiology and Molecular Genetics, School of Medicine, University of California at Irvine, Irvine, California 92697, USA.
RNA. 2009 Jul;15(7):1322-37. doi: 10.1261/rna.1538809. Epub 2009 May 22.
Expression of mitochondrial genomes in Kinetoplastida protists requires massive uracil insertion/deletion mRNA editing. The cascade of editing reactions is accomplished by a multiprotein complex, the 20S editosome, and is directed by trans-acting guide RNAs. Two distinct RNA terminal uridylyl transferases (TUTases), RNA Editing TUTase 1 (RET1) and RNA Editing TUTase 2 (RET2), catalyze 3' uridylylation of guide RNAs and U-insertions into the mRNAs, respectively. RET1 is also involved in mitochondrial mRNA turnover and participates in numerous heterogeneous complexes; RET2 is an integral part of the 20S editosome, in which it forms a U-insertion subcomplex with zinc finger protein MP81 and RNA editing ligase REL2. Here we report the identification of a third mitochondrial TUTase from Trypanosoma brucei. The mitochondrial editosome-like complex associated TUTase (MEAT1) interacts with a 20S editosome-like particle, effectively substituting the U-insertion subcomplex. MEAT1 and RET2 are mutually exclusive in their respective complexes, which otherwise share several components. Similarly to RET2, MEAT1 is exclusively U-specific in vitro and is active on gapped double-stranded RNA resembling editing substrates. However, MEAT1 does not require a 5' phosphate group on the 3' mRNA cleavage fragment produced by editing endonucleases. The functional RNAi complementation experiments showed that MEAT1 is essential for viability of bloodstream and insect parasite forms. The growth inhibition phenotype in the latter can be rescued by coexpressing an RNAi-resistant gene with double-stranded RNA targeting the endogenous transcript. However, preliminary RNA analysis revealed no gross effects on RNA editing in MEAT1-depleted cells and indicated its possible role in regulating the mitochondrial RNA stability.
动质体原生生物中线粒体基因组的表达需要大量尿嘧啶插入/缺失的mRNA编辑。编辑反应的级联由一种多蛋白复合物——20S编辑体完成，并由反式作用引导RNA指导。两种不同的RNA末端尿苷酰转移酶（TUTase），即RNA编辑TUTase 1（RET1）和RNA编辑TUTase 2（RET2），分别催化引导RNA的3'尿苷酰化以及将尿苷插入mRNA中。RET1还参与线粒体mRNA的周转，并参与众多异源复合物；RET2是20S编辑体的一个组成部分，在其中它与锌指蛋白MP81和RNA编辑连接酶REL2形成一个尿苷插入亚复合物。在此，我们报告了从布氏锥虫中鉴定出的第三种线粒体TUTase。线粒体编辑体样复合物相关TUTase（MEAT1）与一个20S编辑体样颗粒相互作用，有效地替代了尿苷插入亚复合物。MEAT1和RET2在各自的复合物中相互排斥，否则它们会共享几个组分。与RET2类似，MEAT1在体外仅对尿苷具有特异性，并且对类似于编辑底物的缺口双链RNA具有活性。然而，MEAT1不需要编辑内切酶产生的3'mRNA切割片段上有5'磷酸基团。功能性RNAi互补实验表明，MEAT1对于血流型和昆虫寄生型寄生虫的生存能力至关重要。通过共表达一个抗RNAi基因和靶向内源性转录本的双链RNA，可以挽救后者的生长抑制表型。然而，初步的RNA分析显示，在MEAT1缺失的细胞中对RNA编辑没有明显影响，并表明其可能在调节线粒体RNA稳定性中发挥作用。
