Structures and Functions of RNA Editing TUTases

 描述(申请人提供)：由布氏锥虫引起的疾病。对许多处于不利地位的国家来说是健康危害。锥虫在进化早期分化，具有独特的RNA加工途径，如线粒体内部U-插入/缺失mRNA编辑和普遍存在的3‘端尿苷化。尿苷化在线粒体RNA代谢中的主要作用促进了对第一末端尿苷转移酶(TUTase)RET1的鉴定。对TUTase的研究进展揭示了它们在真核细胞中广泛的系统发育分布、多样化的结构和深刻的转录组塑造作用。在这个建议中，我们将早期发现的针对所有RNA类的RET1扩展到尿苷基化驱动的RNA加工和衰退的机制研究中。在初步研究中，我们发现RET1 TUTase、DSS1 3‘-5’外切酶和三种缺乏可识别基序的蛋白质构成了一个核心加工复合体，称为线粒体3‘过程组(MPome)。我们表明，尿苷酸靶向降解很可能与反义转录一起作用，以定义成熟引导RNA的3‘边界。最后，我们确定了具有结合底物的RET1的高分辨晶体结构。拟议的实验将解决长期存在的问题，即初级线粒体转录产物在进入编辑途径之前是如何处理的，以及尿苷基化如何控制具有翻译能力的mRNAs的丰度。目标1将定义MPome的分子结构。一种混合结构生物学方法将包括化学交联辅助质谱学(XL-MS)和低温电子显微镜(CryoEM)。我们将建立一个结合了RNA底物的近原子分辨率的络合物模型。目的2将确定3‘尿苷基化靶标引导RNA进行处理的机制。我们认为尿苷偶联、反义转录控制的3‘-5’RNA降解是初级转录本的主要加工途径，并将重点放在gRNA的生物发生上。我们将在体内鉴定MPome结合部位，绘制初级转录本的位置图，并在体外重建加工反应。目的3探讨mRNA5‘修饰与3’5‘降解的关系。成熟的上环转录本(rRNAs和mRNAs)的5‘末端是单磷酸化的，而小环编码的gRNAs保留了转录起始点特有的三磷酸。我们提出了NUDIX水解酶MERS1识别初级上颌骨转录本的5‘端以去除焦磷酸的证据，并提出了一种将mRNA的5’修饰与3‘-5’外核溶解衰变联系起来的机制。这一目标将研究MERS1靶向的特异性以及与MPome的功能相互作用。AIM 4将通过分子动力学辅助筛选虚拟文库来开发RET1的小分子抑制剂。我们将应用化学探针来补充在AIMS 2和3中开发的遗传方法。虽然这个目标的主要目标是寻找化学击倒工具，但以多功能酶为靶点的好处是显而易见的，而且将这些进展转化为未来治疗的可能性很高。

 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.