Guide RNA Binding Complex

摘要 布氏锥虫物种对最边缘化的人造成健康危害和经济困难 世界人口。一些研究得最好的挖洞动物，锥虫也代表了重要的模式， 许多研究领域，包括抗原变异，宿主-病原体相互作用，发育重编程 和线粒体生物学。该项目将阐明大分子RNA编辑的机制， 底物结合复合物（RESC）稳定并将线粒体前mRNA和引导RNA递送到线粒体中。 U-插入/缺失编辑途径，并协调编辑的mRNA的多聚腺苷酸化和翻译。我们 建立RESC平台作为编辑全酶的RNA结合组分， 它在编辑反应中的作用，以及RNA编辑过程之外的功能。为此，我们证明， RESC相关MERS 1焦磷酸水解酶和KPAP 1 poly（A）聚合酶靶向前mRNA 5′和3′ 分别结束。重要的是，5′焦磷酸盐去除和3′ A加尾似乎对前体mRNA的表达至关重要。 编辑前的稳定性。相反，建议RESC中的特定模块将 3′ A/U加尾编辑和mRNA与核糖体的结合。总的来说，现有的证据 将~25多肽RESC复合物定位为线粒体RNA加工的多模式联系。 此外，初步研究RESC相关的MERS 1复合物，RNA聚合酶（MTRNAP），和3′端 加工体（MPsome）挑战了长期存在的多顺反子大环转录模型， 初级转录物的核酸内切分配。这些实验将加深对RNA的理解 通过以近原子分辨率确定RESC结构和个体的RNA结合特异性来编辑 亚单位。我们将测试一个广泛的功能假设，即离散的RESC模块协调mRNA的完成， 3′修饰和翻译激活的编辑。最后，我们提出了一个全新的概念， 单顺反子前体mRNA，其从单个启动子转录并通过5 ′端修饰形成， 反义RNA控制的3′-5′降解。通过阐明RESC的结构、RNA结合特性和 高阶的相互作用，并评估范式转移“单顺反子假说”，这个计划将 扩大关键寄生虫的特定过程的知识，并可能提供新的药物靶点。

ABSTRACT Trypanosoma brucei species inflict health hazards and economic hardship on arguably the most marginalized populations in the world. Some of the best-studied Excavata, trypanosomes also represent important models in many areas of research, including antigenic variation, host-pathogen interaction, developmental reprogramming and mitochondrial biology. This project will elucidate mechanisms by which macromolecular RNA editing substrate binding complex (RESC) stabilizes and delivers mitochondrial pre-mRNAs and guide RNAs into the U-insertion/deletion editing pathway, and coordinates polyadenylation and translation of edited mRNAs. We establish the RESC platform as the RNA binding constituent of the editing holoenzyme and seek to investigate its role in editing reactions, and functions beyond the RNA editing process. To this end, we demonstrate that RESC-associated MERS1 pyrophosphohydrolase and KPAP1 poly(A) polymerase target pre-mRNA 5′ and 3′ ends, respectively. Importantly, both 5′ pyrophosphate removal and 3′ A-tailing appear to be critical for pre-mRNA stabilization prior to editing. Conversely, specific module within RESC is suggested to couple the completion of editing with post-editing 3′ A/U-tailing and mRNA binding to the ribosome. Collectively, the existing evidence positions the ~25 polypeptide RESC complex as the multimodal nexus of mitochondrial RNA processing. Furthermore, initial investigation of RESC-associated MERS1 complex, RNA polymerase (MTRNAP), and the 3′ processome (MPsome) challenges the long-standing model of multicistronic maxicircle transcription and endonucleolytic partitioning of primary transcripts. The proposed experiments will deepen understanding of RNA editing by determining the RESC structure at near-atomic resolution and RNA binding specificities of individual subunits. We will test a broad functional hypothesis that discrete RESC modules coordinate completion of mRNA editing with 3′ modification and translational activation. Finally, we put forward a fundamentally novel concept of monocistronic pre-mRNAs that are transcribed from individual promoters and shaped by 5ʹ modification and antisense RNA-controlled 3′-5′ degradation. By elucidating the RESC structure, RNA binding properties, and higher-order interactions, and evaluating the paradigm-shifting “monocistronic hypothesis,” this program will expand the knowledge of critical parasite-specific processes and may provide new drug targets.