Structures and Functions of RNA Editing TUTases

 描述（由申请方提供）：布氏锥虫引起的疾病。对许多贫困国家来说是健康危害。锥虫在进化的早期就发生了分化，并具有独特的RNA加工途径，如线粒体内部的U插入/缺失mRNA编辑和普遍存在的3′端尿苷酸化。尿苷酸化在线粒体RNA代谢中的主要作用使第一末端尿苷酰转移酶（TUTase）RET 1的鉴定充满热情。对TUTases的深入研究揭示了其在真核细胞中广泛的系统发育分布、多样的结构和深刻的转录组形成作用。在这个提议中，我们将早期发现的靶向所有RNA类别的RET 1扩展到尿苷化驱动的RNA加工和衰变的机制研究。在前期的研究中，我们发现RET 1 TUTase、DSS 1 3′-5′核酸外切酶和三种缺乏可识别基序的蛋白质组成了一个核心加工复合体，称为线粒体3′加工体（MPsome）。我们发现，尿苷酸化靶向降解最有可能与反义转录协同作用，以定义成熟向导RNA的3′边界。最后，我们确定了一个高分辨率的晶体结构与绑定基板的RET 1。拟议的实验将解决长期存在的问题，即初级线粒体转录本在进入编辑途径之前如何加工，以及尿苷酸化如何控制具有免疫活性的mRNA的丰度。目的1将定义MPsome的分子结构。混合结构生物学方法将包括化学交联辅助质谱（XL-MS）和冷冻电子显微镜（cryoEM）。我们将建立一个近原子分辨率的复合物模型与结合RNA底物。目的2将确定3′尿苷化靶向指导RNA进行加工的机制。我们认为，尿苷酸化偶联的反义转录控制的3′-5′ RNA降解代表了初级转录物的主要加工途径，并将关注gRNA生物合成。我们将确定在体内MPsome结合位点，地图的初级转录本的位置和重建在体外加工反应。目的3探讨mRNA 5′端修饰与3′端5′端降解的关系。成熟的大环转录物（rRNA和mRNA）的5′末端被单磷酸化，而小环编码的gRNA保留转录起始位点的三磷酸特征。我们提出NUDIX水解酶MERS 1识别初级大环转录物的5′末端以去除焦磷酸的证据，并提出了一种连接mRNA的5′修饰和3′-5′核酸外切降解的机制。该目标将研究MERS 1靶向的特异性以及与MPsome的功能相互作用。目的4利用分子动力学技术辅助虚拟文库筛选RET 1小分子抑制剂。我们将应用化学探针来补充目标2和3中开发的遗传方法。虽然该目标的主要目标是寻找化学敲除工具，但靶向多功能酶的好处是显而易见的，并且将这些进展转化为未来疗法的可能性很高。

 DESCRIPTION (provided by applicant): Diseases caused by Trypanosoma brucei spp. represent health hazards for many disadvantaged countries. Trypanosomatids diverged early in evolution and possess unique RNA processing pathways, such as mitochondrial internal U-insertion/deletion mRNA editing and ubiquitous 3′ end uridylation. The principal role of uridylation in mitochondrial RNA metabolism enthused identification of the first terminal uridyltransferase (TUTase) RET1. Progressing studies of TUTases uncovered their wide phylogenetic distribution, diverse structures and profound transcriptome-shaping roles in eukaryotic cells. In this proposal, we expand earlier finding of RET1 targeting all RNA classes into a mechanistic investigation of uridylation-driven RNA processing and decay. In preliminary studies, we discovered that RET1 TUTase, DSS1 3′-5′ exonuclease and three proteins lacking recognizable motifs constitute a core processing complex termed the mitochondrial 3′ processome (MPsome). We show that uridylation-targeted degradation most likely operates in concert with antisense transcription to define 3′ boundaries of mature guide RNAs. Finally, we determined a high-resolution crystal structure of RET1 with bound substrates. The proposed experiments will resolve the long-standing questions of how primary mitochondrial transcripts are processed prior to entering editing pathway and how uridylation controls the abundance of translationally-competent mRNAs. Aim 1 will define the molecular architecture of the MPsome. A hybrid structural biology approach will include chemical crosslinking-assisted mass spectrometry (XL-MS) and cryoelectron microscopy (cryoEM). We will build a near-atomic resolution complex model with bound RNA substrates. Aim 2 will determine the mechanism by which 3′ uridylation targets guide RNAs for processing. We propose that uridylation-coupled, antisense transcription-controlled 3′-5′ RNA degradation represents the major processing pathway of primary transcripts and will focus on gRNA biogenesis. We will identify in vivo MPsome binding sites, map positions of primary transcripts and reconstitute processing reactions in vitro. Aim 3 will investigate the relationship between mRNA 5′ modification and 3′5′ degradation. The 5′ termini of mature maxicircle transcripts (rRNAs and mRNAs) are monophosphorylated while minicircle-encoded gRNAs retain triphosphates characteristic of transcription start site. We present evidence that NUDIX hydrolase MERS1 recognizes 5′ termini of primary maxicircle transcripts to remove pyrophosphate and propose a mechanism linking mRNA's 5′ modification and 3′-5′ exonucleolytic decay. This Aim will investigate the specificty of MERS1 targeting and functional interactions with the MPsome. Aim 4 will develop small-molecule inhibitors of RET1 by molecular dynamics-assisted screening of virtual libraries. We will apply chemical probes to complement genetic approaches developed in Aims 2 and 3. Although this Aim's main objective is to search for chemical knockdown tools, the benefits of targeting a multifunctional enzyme are apparent, and the likelihood of converting such advances into future therapies is high.