Repeat-containing RNA Binding Proteins of Trypanosomes

含有重复序列的锥虫 RNA 结合蛋白

• 批准号: 9187995
• 负责人: Inna Afasizheva
• 金额: $ 40.93万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-05 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9187995
• 关键词: Address; African Trypanosomiasis; Amino Acids; Animal Model; Appearance; Area; Binding; Biochemical; Biochemistry; Biogenesis; Biological Assay; Biology; Code; Complex; Country; Crystallography; DNA Structure; Deposition; Disadvantaged; Disease; Drug resistance; Due Process; Ensure; Enzymes; Event; Family; Gene Expression; Genetic; Genome; Goals; Guide RNA; Health Hazards; Helix-Turn-Helix Motifs; Human; Investigation; Mass Spectrum Analysis; Mastigophora; Mediating; Messenger RNA; Mitochondria; Mitochondrial RNA; Modification; Molecular; Molecular Biology; Monitor; Nature; Nuclear; Open Reading Frames; Organism; Parasites; Parasitology; Pathway interactions; Phosphodiesterase I; Polyadenylation; Polynucleotide Adenylyltransferase; Process; Protein Engineering; Proteins; Proteomics; Publishing; RNA; RNA Binding; RNA Editing; RNA Processing; RNA Sequences; RNA-Binding Proteins; Reaction; Recruitment Activity; Repression; Research; Ribosomal RNA; Ribosomes; Role; Shapes; Small RNA; Source; Specificity; Structure; Tail; Testing; Therapeutic; Therapeutic Intervention; Transcript; Translations; Trypanosoma; Trypanosoma brucei brucei; Work; base; blocking factor; crosslink; deep sequencing; health economics; in vivo; insertion/deletion mutation; mRNA Decay; mRNA Precursor; mRNA Stability; member; new therapeutic target; public health relevance; resistant strain; transcriptome; transcriptome sequencing; vector control

 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.

 描述（由申请人提供）：由布氏锥虫引起的疾病。对许多弱势国家的健康构成危害。由于病媒控制失败和耐药性上升，非洲昏睡病的再次出现需要寻找新的药物靶点，重点关注寄生虫特异性过程。锥虫线粒体 RNA 加工途径与人类极其不同，代表了治疗干预点的潜在来源。 mRNA 编辑已被广泛研究，但编辑前和编辑后处理事件的重要性直到最近才出现。我们发现了线粒体聚腺苷酸聚合酶 (KPAP1) 和末端尿苷酰转移酶 (RET1)，并表明相互交织的聚腺苷酸化和尿苷酸化过程对于 mRNA 稳定性、翻译和衰变至关重要。在这些研究中，我遇到了五肽（PPR，35 个氨基酸）螺旋重复的不同家族的 RNA 结合蛋白，它们填充聚腺苷酸化和翻译复合物。通过关注 mRNA 3' 末端的定义、修饰和稳定性，该提案引入 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/尿苷化机制。我假设 3' 修饰的两种模式，即预编辑添加短 A 尾和后编辑 A/U 尾，分别服务于 mRNA 稳定和翻译承诺的不同目的。 2)确定基于尿苷化的mRNA衰减原理。将测试 RET1 TUTase 催化的尿苷化加速 mRNA 3'-5' 降解的假设。我还假设大多数转录本都通过一种特定的 PPR 蛋白来稳定，这种蛋白可以阻止 mRNA 尿苷化。 3) 阐明假定的 mRNA 3' 定义和稳定因子的功能。我建议在聚腺苷酸化之前将 PPR 因子沉积到前 mRNA 上以定义 3' 末端。我们专注于锥虫中的精选 PPR 蛋白，但长期目标是阐明包含重复蛋白的 RNA 识别机制，这对于 RNA 加工和线粒体生物学领域至关重要。