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首页> 外文期刊>Journal of Molecular Biology >Mechanism of U insertion RNA editing in trypanosome mitochondria: the bimodal TUTase activity of the core complex.
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Mechanism of U insertion RNA editing in trypanosome mitochondria: the bimodal TUTase activity of the core complex.

机译：锥虫线粒体中U插入RNA编辑的机制：核心复合物的双峰TUTase活性。

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摘要

Expression of the trypanosomal mitochondrial genome requires the insertion and deletion of uridylyl residues at specific sites in pre-mRNAs. RET2 terminal uridylyl transferase is an integral component of the RNA editing core complex (RECC) and is responsible for the guide-RNA-dependent U insertion reaction. By analyzing RNA-interference-based knock-in Trypanosoma brucei cell lines, purified editing complex, and individual protein, we have investigated RET2's association with the RECC. In addition, the U insertion activity exhibited by RET2 as an RECC subunit was compared with characteristics of the monomeric protein. We show that interaction of RET2 with RECC is accomplished via a protein-protein contact between its middle domain and a structural subunit, MP81. The recombinant RET2 catalyzes a faithful editing on gapped (precleaved) double-stranded RNA substrates, and this reaction requires an internal monophosphate group at the 5' end of the mRNA 3' cleavage fragment. However, RET2 processivity is limited to insertion of three Us. Incorporation into the RECC voids the internal phosphate requirement and allows filling of longer gaps similar to those observed in vivo. Remarkably, monomeric and RECC-embedded enzymes display a similar bimodal activity: the distributive insertion of a single uracil is followed by a processive extension limited by the number of guiding nucleotides. Based on the RNA substrate specificity of RET2 and the purine-rich nature of U insertion sites, we propose that the distributive +1 insertion creates a substrate for the processive gap-filling reaction. Upon base-pairing of the +1 extended 5' cleavage fragment with a guiding nucleotide, this substrate is recognized by RET2 in a different mode compared to the product of the initial nucleolytic cleavage. Therefore, RET2 distinguishes base pairs in gapped RNA substrates which may constitute an additional checkpoint contributing to overall fidelity of the editing process.
机译：锥虫线粒体基因组的表达需要在前mRNA的特定位点插入和删除尿嘧啶残基。 RET2末端尿嘧啶转移酶是RNA编辑核心复合体（RECC）的组成部分，负责引导RNA依赖性的U插入反应。通过分析基于RNA干扰的敲入布鲁氏锥虫细胞系，纯化的编辑复合体和单个蛋白质，我们研究了RET2与RECC的关联。另外，将RET2作为RECC亚单位表现出的U插入活性与单体蛋白的特性进行了比较。我们显示，RET2与RECC的相互作用是通过其中间结构域与结构亚基MP81之间的蛋白质-蛋白质接触来实现的。重组RET2催化对空缺的（预切割的）双链RNA底物的忠实编辑，并且此反应需要在mRNA 3'裂解片段的5'端带有一个内部单磷酸基团。但是，RET2的合成能力仅限于插入三个Us。掺入RECC可以消除内部磷酸盐的需求，并可以填补与体内观察到的相似的更长的缺口。值得注意的是，单体酶和嵌入RECC的酶表现出相似的双峰活性：单个尿嘧啶的分布插入后，是一个连续的延伸，该延伸受到指导核苷酸数目的限制。基于RET2的RNA底物特异性和U插入位点的富嘌呤性质，我们建议+1分布插入为进行性间隙填充反应创建底物。与引导核苷酸进行+1延伸的5'裂解片段的碱基配对后，该底物被RET2识别，其模式与初始核酸裂解产物相比有所不同。因此，RET2区分了空缺的RNA底物中的碱基对，这可能构成有助于编辑过程整体保真度的附加检查点。

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