Decoding RNA-Protein Interactions in Trypanosoma Telomerase

解码锥虫端粒酶中 RNA-蛋白质相互作用

基本信息

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
项目类别：
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

项目摘要

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.

项目概要端粒酶是一种独特的核糖核蛋白酶，可不断添加端粒重复序列，从其复制整合RNA成分，位于线性染色体的末端，以防止真核生物中基因组的不稳定。这该提案旨在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（Trypanosoma brucei）的调控，一种早期分化的寄生原生生物，通过多种方式增殖人类和昆虫在形态上不同的生命周期形式。在布氏锥虫中，端粒结构起着在抗原变异的调节中发挥重要作用，使寄生虫能够建立长期感染。特别是，极短的端粒可能会危害端粒的完整性及其（亚）端粒的稳定性。毒力基因和寄生虫的生存。因此，了解控制端粒的机制 T. brucei 中的复制可以为控制寄生虫增殖提供重要线索。端粒酶是主要布氏锥虫端粒合成机制。两个高度保守的端粒酶RNA结构域， RNA 模板和 eCR4/5 独立结合催化蛋白端粒酶逆转录酶 (TERT) 在端粒合成过程中，是体外催化重建所需的唯一 RNA 元件活性端粒酶。然而，T.brucei 端粒酶 RNA 在与纤毛虫、酵母和脊椎动物端粒酶 RNA 相比，上述结构域具有新的结构域，这表明端粒酶的新模式端粒合成的调控。因此，我们的假设是这些不寻常的序列和结构布氏锥虫端粒酶 RNA 结构域的多样性导致 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 T. brucei 两个复制阶段的结构探测数据表明 RNA 折叠和端粒酶激活可以在发育过程中受到调节。了解特定阶段的结构重组是如何进行的和 RNA-蛋白质相互作用控制 T. brucei 中的端粒酶调节，在该提案的目标 1 中，我们将确定 T. brucei 中上述两个域中 RNA-蛋白质相互作用的分子要求端粒酶。该提案的目标 2 将通过剖析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。综上所述，这研究将为PI确定端粒酶激活核心成分的长期目标奠定基础以及临床上重要的原生生物中端粒长度稳态和基因组完整性的相互作用。