Madina Bhaskara R, Kumar Vikas, Mooers Blaine H M, Cruz-Reyes Jorge
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, United States of America.
Department of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, United States of America; Department of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, United States of America.
PLoS One. 2015 Apr 30;10(4):e0123441. doi: 10.1371/journal.pone.0123441. eCollection 2015.
Adaptation and survival of Trypanosoma brucei requires editing of mitochondrial mRNA by uridylate (U) insertion and deletion. Hundreds of small guide RNAs (gRNAs) direct the mRNA editing at over 3,000 sites. RNA editing is controlled during the life cycle but the regulation of substrate and stage specificity remains unknown. Editing progresses in the 3' to 5' direction along the pre-mRNA in blocks, each targeted by a unique gRNA. A critical editing factor is the mitochondrial RNA binding complex 1 (MRB1) that binds gRNA and transiently interacts with the catalytic RNA editing core complex (RECC). MRB1 is a large and dynamic complex that appears to be comprised of distinct but related subcomplexes (termed here MRBs). MRBs seem to share a 'core' complex of proteins but differ in the composition of the 'variable' proteins. Since some proteins associate transiently the MRBs remain imprecisely defined. MRB1 controls editing by unknown mechanisms, and the functional relevance of the different MRBs is unclear. We previously identified two distinct MRBs, and showed that they carry mRNAs that undergo editing. We proposed that editing takes place in the MRBs because MRBs stably associate with mRNA and gRNA but only transiently interact with RECC, which is RNA free. Here, we identify the first specialized functions in MRBs: 1) 3010-MRB is a major scaffold for RNA editing, and 2) REH2-MRB contains a critical trans-acting RNA helicase (REH2) that affects multiple steps of editing function in 3010-MRB. These trans effects of the REH2 include loading of unedited mRNA and editing in the first block and in subsequent blocks as editing progresses. REH2 binds its own MRB via RNA, and conserved domains in REH2 were critical for REH2 to associate with the RNA and protein components of its MRB. Importantly, REH2 associates with a ~30 kDa RNA-binding protein in a novel ~15S subcomplex in RNA-depleted mitochondria. We use these new results to update our model of MRB function and organization.
布氏锥虫的适应与存活需要通过尿苷酸（U）的插入和缺失来编辑线粒体mRNA。数百个小向导RNA（gRNA）指导着超过3000个位点的mRNA编辑。RNA编辑在生命周期中受到调控，但底物和阶段特异性的调控机制仍不清楚。编辑沿着前体mRNA从3'端向5'端以块的形式进行，每个块由一个独特的gRNA靶向。一个关键的编辑因子是线粒体RNA结合复合体1（MRB1），它结合gRNA并与催化性RNA编辑核心复合体（RECC）短暂相互作用。MRB1是一个庞大且动态的复合体，似乎由不同但相关的亚复合体（在此称为MRBs）组成。MRBs似乎共享一个蛋白质“核心”复合体，但“可变”蛋白质的组成不同。由于一些蛋白质是短暂结合的，MRBs的定义仍不精确。MRB1通过未知机制控制编辑，不同MRBs的功能相关性尚不清楚。我们之前鉴定出了两种不同的MRBs，并表明它们携带经过编辑的mRNA。我们提出编辑发生在MRBs中，因为MRBs与mRNA和gRNA稳定结合，但仅与无RNA的RECC短暂相互作用。在此，我们确定了MRBs中的首个特殊功能：1）3010-MRB是RNA编辑的主要支架，2）REH2-MRB包含一个关键的反式作用RNA解旋酶（REH2），它影响3010-MRB中编辑功能的多个步骤。REH2的这些反式作用包括未编辑mRNA的加载以及随着编辑进展在第一个块和后续块中的编辑。REH2通过RNA与自身的MRB结合，并且REH2中的保守结构域对于REH2与MRB的RNA和蛋白质成分结合至关重要。重要的是，REH2在RNA耗尽的线粒体中与一个约30 kDa的RNA结合蛋白在一个新的约15S亚复合体中结合。我们利用这些新结果更新了我们关于MRB功能和组织的模型。
