REPEAT-CONTAINING RNA BINDING PROTEINS OF TRYPANOSOMES

摘要 鞭毛动质体原生动物主要对人类造成公共卫生危害和经济负担， 发展中国家的边缘化人口。非洲人类和动物锥虫的病原体， 布氏锥虫，导致一些研究最多，但最难管理和治愈的寄生虫感染。 病媒控制失败、缺乏疫苗和毒性治疗方法，使鉴定、分子生物学和生物学方面的责任 了解和验证新药靶点。从基础科学的角度来看，这些早期 分支挖掘代表了线粒体RNA生物学，抗原变异，宿主-病原体 相互作用、转录后调节和其他过程。上一个供资期间的主要调查结果 定位RNA结合五肽重复（PPR，35个氨基酸）蛋白家族作为中心导管 控制线粒体基因表达。螺旋-转角-螺旋重复序列组装成 超螺旋阵列识别延伸的和组成多样的RNA序列， 超过40个PPR的角色。通常包括锥虫特异性结构域，这些RNA "序列阅读器" 将其他非特异性修饰酶提交给其底物，或阻断RNA降解， 扩展名.本项目将阐明核编码的PPRs控制线粒体的机制 mRNA生物合成和翻译通过招募酶复合物和核糖体转录在定义的 加工阶段。我们建立了三个优先领域，重点是PPR，使mRNA 5 ′端修饰， 3 ′腺苷酸化/尿苷酸化和翻译。基于我们最近发现的基因特异性转录 启动和核酸外切酶前mRNA加工，我们确定了PPR因子作为"蛋白帽"，"编辑"， 传感器、聚腺苷酸结合蛋白和潜在的翻译激活剂。我们的目标是剖析 整合这些功能的机制，并通过含有重复序列的RNA识别来破译RNA识别的原理。 proteins.通过阐明它们的RNA结合位点、结构、相互作用和遗传改变的结果， 蛋白质工程，该计划建立在我们的势头在一个重要的领域，并扩大了知识， 关键的病原体特异性过程。

ABSTRACT Flagellated kinetoplastid protozoans inflict public health hazards and economic burden predominately on marginalized populations in the developing world. The agents of African human and animal trypanosomiasis, Trypanosoma brucei sp., cause some of the most-studied, but least manageable and curable parasitic infections. Failing vector control, lack of vaccines and toxic therapeutics place the onus on identification, molecular understanding and validation of new drug targets. From the fundamental science perspective, these early branching Excavata represent valuable models of mitochondrial RNA biology, antigenic variation, host-pathogen interaction, post-transcriptional regulation, and other processes. Major findings in the previous funding period position a family of RNA binding pentatricopeptide repeat (PPR, 35 amino acids) proteins as the central conduit controlling mitochondrial gene expression. The unique ability of helix-turn-helix repeats to assemble into superhelical arrays recognizing extended and compositionally diverse RNA sequences enables the essential roles of more than 40 PPRs. Often including trypanosome-specific domains, these RNA “sequence readers” commit otherwise non-specific modification enzymes to their substrates, or block RNA degradation and extension. This project will elucidate the mechanisms by which nuclear-encoded PPRs govern mitochondrial mRNA biogenesis and translation via recruiting enzymatic complexes and ribosomes to transcripts at defined processing stages. We establish three priority areas focusing on PPRs that enable mRNA 5′ end modification, 3′ adenylation/uridylation, and translation. Building on our recent discoveries of gene-specific transcription initiation and exonucleolytic pre-mRNA processing, we identified PPR factors acting as “protein cap,” “editing sensor,” poly(A) binding protein, and potential translational activators. Our goal is to dissect the molecular machinery integrating these functions and to decipher principles of RNA recognition by repeat-containing proteins. By elucidating their RNA binding sites, structures, interactions, and outcomes of genetic alteration and protein engineering, this program builds on our momentum in a significant field and expands the knowledge of critical pathogen-specific processes.