Repeat-containing RNA Binding Proteins of Trypanosomes

 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。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标，重点是寄生虫特有的过程。锥虫线粒体RNA处理途径与人类极为不同，是治疗干预点的潜在来源。人们对信使核糖核酸的编辑进行了广泛的研究，但编辑前后加工事件的意义直到最近才开始显现。我们发现了线粒体多聚酶(KPAP1)和末端尿苷转移酶(RET1)，并表明相互交织的多聚腺苷化和尿苷化过程对mRNA的稳定性、翻译和腐烂至关重要。在这些研究中，我遇到了不同的五肽(PPR，35个氨基酸)螺旋重复RNA结合蛋白家族，它们存在于多聚腺苷化和翻译复合体中。通过关注mRNA3的末端定义、修饰和稳定性，本建议引入PPR蛋白作为线粒体基因表达的关键调节蛋白。实验方法涉及生物化学、蛋白质组学、结晶学、遗传学和深RNA测序。该建议的目的是：1)剖析一般的和转录本特异的mRNA腺化/尿化机制。我假设3‘修饰的两种模式，即编辑前添加短A尾巴和编辑后添加A/U尾巴，分别服务于mRNA稳定和致力于翻译的目的。2)确定基于尿苷基化的信使核糖核酸的衰变原理。一种假设，即RET1 TUTase催化的尿苷基化加速了mRNA3‘-5’的降解，这一假设将得到验证。我还假设，大多数转录本都是由一种特定的PPR蛋白稳定下来的，这种蛋白可以阻止mRNA的尿化。3)阐明一个可能的mRNA3‘定义和稳定因子的功能。我建议在多聚腺苷化之前，将PPR因子沉积在前mRNA上，以定义3‘端。我们的重点是选择锥虫体内的PPR蛋白，但长期目标是阐明含有重复序列的蛋白识别RNA的机制，这将对RNA加工和线粒体生物学领域具有重要意义。

 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.