Repeat-containing RNA Binding Proteins of Trypanosomes

 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。由于病媒控制失败和抗药性上升，非洲昏睡病的重新出现需要寻找新的药物靶点，重点是寄生虫特异性过程。锥虫线粒体RNA加工途径与人类极为不同，代表了治疗干预点的潜在来源。mRNA编辑已经得到了广泛的研究，但编辑前和编辑后处理事件的重要性只是最近才出现。我们发现了线粒体多聚腺苷酸聚合酶（KPAP 1）和末端尿苷酰转移酶（RET 1），并表明交织的多聚腺苷酸化和尿苷酸化过程对mRNA的稳定性，翻译和衰变至关重要。在这些研究中，我遇到了一个不同的家庭五肽（PPR，35个氨基酸）螺旋重复含有RNA结合蛋白，填充聚腺苷酸化和翻译复合物。通过关注mRNA 3'端的定义、修饰和稳定性，该提议引入PPR蛋白作为线粒体基因表达的关键调节因子。实验方法涉及生物化学，蛋白质组学，晶体学，遗传学和深度RNA测序。该提案旨在：1）剖析一般和转录特异性mRNA腺苷酸化/尿苷酸化机制。我假设3'端修饰的两种模式，即编辑前添加短A尾和编辑后添加A/U尾，分别用于mRNA稳定化和翻译承诺的离散目的。2)确定基于尿苷酸化的mRNA衰变的原理.将检验RET 1 TUTase催化的尿苷化加速mRNA 3 '-5'降解的假设。我还认为，大多数转录稳定的特定PPR蛋白阻断mRNA尿苷酸化。3)阐明一个假定的mRNA 3'端定义和稳定因子的功能。我建议在聚腺苷酸化之前将PPR因子沉积在前mRNA上以确定3'端。我们的重点是选择锥虫中的PPR蛋白，但长期目标是阐明含重复序列的蛋白识别RNA的机制，这将是RNA加工和线粒体生物学领域的根本重要性。

 DESCRIPTION (provided by applicant): Diseases caused by Trypanosoma brucei spp. represent health hazards for many disadvantaged countries. Because of failing vector control and the rise of drug-resistance, the re-appearance of African sleeping sickness necessitates a search for new drug targets with emphasis on parasite-specific processes. Trypanosomal mitochondrial RNA processing pathways are extremely divergent from those of humans and represent a potential source of therapeutic intervention points. The mRNA editing has been extensively studied, but the significance of pre- and post-editing processing events emerged only recently. We discovered the mitochondrial poly (A) polymerase (KPAP1) and terminal uridyltransferase (RET1), and showed that intertwined polyadenylation and uridylation processes are critical for mRNA stability, translation and decay. In these studies, I encountered a diverse family of pentatricopeptide (PPR, 35 amino acids) helical repeat- containing RNA binding proteins that populate polyadenylation and translation complexes. By focusing on mRNA 3' end definition, modification and stability, this proposal introduces PPR proteins as key regulators of mitochondrial gene expression. The experimental approaches involve biochemistry, proteomics, crystallography, genetics and deep RNA sequencing. The proposal aims to: 1) Dissect general and transcript-specific mRNA adenylation/uridylation mechanisms. I hypothesize that the two modes of 3' modification, pre-editing addition of a short A-tail and post-editing A/U-tailing, serve discrete purposes of mRNA stabilization and commitment to translation, respectively. 2) Determine principles of uridylation- based mRNA decay. A hypothesis that RET1 TUTase-catalyzed uridylation accelerates mRNA 3'-5' degradation will be tested. I also posit that most transcripts are stabilized by a specific PPR protein that blocks mRNA uridylation. 3) Elucidate functions of a putative mRNA 3' definition and stabilization factor. I suggest that a PPR factor is deposited onto the pre-mRNA prior to polyadenylation to define the 3' end. We focus on select PPR proteins in trypanosomes, but the long term goal is to illuminate the mechanisms of RNA recognition by repeat-containing proteins, which will be of fundamental importance for RNA processing and mitochondrial biology fields.