Decoding RNA-Protein Interactions in Trypanosoma Telomerase

解码锥虫端粒酶中 RNA-蛋白质相互作用

• 批准号: 10515146
• 负责人: Kausik Chakrabarti
• 金额: $ 47.44万
• 依托单位: UNIVERSITY OF NORTH CAROLINA CHARLOTTE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10515146
• 关键词: Active Sites; Adopted; Affect; Antigenic Variation; Binding; Biochemical; Biology; Cell Proliferation; Cell Shape; Cells; Chromosomes; Clinical; Complex; DNA; Data; Detection; Development; Disease; Elements; Enzymes; Eukaryota; Foundations; Gene Expression; Genes; Genomic Instability; Goals; Heterogeneous-Nuclear Ribonucleoprotein Group F-H; Heterogeneous-Nuclear Ribonucleoproteins; Holoenzymes; Homeostasis; Human; Immune; In Vitro; Infection; Insecta; Length; Life; Life Cycle Stages; Mass Spectrum Analysis; Mediating; Modeling; Modification; Molecular; Molecular Chaperones; Molecular Conformation; Morphology; Multienzyme Complexes; Mutation; Neurologic; Nuclear; Nuclear Proteins; Nucleotides; Parasite Control; Parasites; Pathogenesis; Pathway interactions; Play; Proliferating; Protein Family; Proteins; Proteomics; RNA; RNA Folding; RNA Stability; RNA-Directed DNA Polymerase; RNA-Protein Interaction; Regulation; Research; Ribonucleoproteins; Role; Site; Structure; Telomerase; Telomerase RNA Component; Telomere Maintenance; Trypanosoma; Trypanosoma brucei brucei; Tumor stage; Virulence; Yeasts; base; genome integrity; novel; pathogen; prevent; reconstitution; response; scaffold; telomere

Project Summary Telomerase is a unique ribonucleoprotein enzyme that processively adds telomeric repeats, copied from its integral RNA component, to the ends of linear chromosomes to prevent genome instability in eukaryotes. This proposal seeks to define RNA folding and RNA-protein interactions that are critically important for telomerase regulation in Trypanosoma brucei, an early divergent parasitic protist that proliferates through multiple morphologically distinct life cycle forms in humans and insects. In T. brucei, the telomere structure plays an important role in regulation of antigenic variation that enables the parasite to establish a long-term infection. Particularly, extremely short telomeres could jeopardize telomere integrity, stability of their (sub)telomeric virulence genes and parasite survival. Therefore, understanding the mechanism that controls telomere replication in T. brucei could provide important clues to control parasite proliferation. Telomerase is the major mechanism of telomere synthesis in T. brucei. Two highly conserved telomerase RNA structural domains, the RNA template and eCR4/5 independently bind the catalytic protein, telomerase reverse transcriptase (TERT) during telomere synthesis and are the only required RNA elements for in vitro reconstitution of catalytically active telomerase. However, T.brucei telomerase RNA has unusual sequence and structural composition in the above domains compared to ciliate, yeast and vertebrate telomerase RNAs, suggesting novel modes of regulation for telomere synthesis. Therefore, our hypothesis is that these unusual sequence and structural diversity of T. brucei telomerase RNA domains cause differences in RNA-protein interactions and conformational changes, resulting in species-specific telomerase assembly and activity. Our recent RNA structure probing data from two replicative stages of T. brucei suggests that RNA folding and telomerase activation could be developmentally regulated. To understand how stage -specific structural rearrangements and RNA-proteins interactions control telomerase regulation in T. brucei, in Aim 1 of the proposal we will determine molecular requirements of RNA-protein interactions in the above two domains in T. brucei telomerase. Aim 2 of this proposal will explore additional requirements for telomerase regulation by dissecting RNA-specific factors that are required for functional telomerase RNP assembly and activity. In summary, this research will lay the foundation for the PI's long-term goal to define core components of telomerase activation and interactions for telomere length homeostasis and genome integrity in a clinically important protist.

项目摘要 端粒酶是一种独特的核糖核蛋白酶，它可以从端粒复制端粒重复序列， 在真核生物中，RNA是一个完整的RNA组分，它可以被连接到线性染色体的末端，以防止基因组的不稳定性。这 一项提案试图定义对端粒酶至关重要的RNA折叠和RNA-蛋白质相互作用 布氏锥虫是一种早期分化的寄生原生生物， 在人类和昆虫的形态学上不同的生命周期形式。于T.布鲁塞，端粒结构起着 在调节抗原变异中发挥重要作用，使寄生虫能够建立长期感染。 特别是，极短的端粒可能会危及端粒的完整性，其（亚）端粒的稳定性， 毒力基因和寄生虫存活。因此，了解控制端粒的机制 T.布氏杆菌可为控制寄生虫增殖提供重要线索。端粒酶是 端粒合成的机制。布鲁塞。两个高度保守的端粒酶RNA结构域， RNA模板和eCR 4/5独立地结合催化蛋白，端粒酶逆转录酶（TERT） 在端粒的合成过程中，是唯一需要的RNA元件在体外重建的催化 活性端粒酶然而，布氏锥虫端粒酶RNA具有不寻常的序列和结构组成， 与纤毛虫、酵母和脊椎动物端粒酶RNA相比， 调节端粒合成。因此，我们的假设是，这些不寻常的序列和结构 多样性T.布鲁氏菌端粒酶RNA结构域引起RNA-蛋白质相互作用的差异， 构象变化，导致物种特异性端粒酶组装和活性。我们最近的RNA 结构探测数据来自T.布氏菌认为RNA折叠和端粒酶 激活可以受到发育调节。为了了解特定阶段的结构重组 RNA-蛋白质相互作用控制T.布鲁塞，在提案的目标1中，我们将 确定T.布氏 端粒酶本提案的目标2将通过解剖来探索端粒酶调控的额外要求。 功能性端粒酶RNP组装和活性所需的RNA特异性因子。总之，这 这项研究将为PI的长期目标奠定基础，以确定端粒酶激活的核心成分 和端粒长度稳态和基因组完整性在临床上重要的原生生物的相互作用。