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Administrative Supplement to Promote Diversity in DecodingRNA-Protein Interactions in Trypanosoma Telomerase

促进锥虫端粒酶 RNA-蛋白质相互作用解码多样性的行政补充

基本信息

批准号：
10797998
负责人：
Kausik Chakrabarti
金额：
$ 13.1万
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：
2023
资助国家：
美国
起止时间：
2023-05-01 至 2025-04-30
项目状态：
未结题

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. The overall goal of this diversity research supplement proposal is to define the role of post-translational modifications of the telomerase catalytic protein component, TERT in RNA-protein interactions and telomerase regulation in Trypanosoma brucei. T. brucei is 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. Telomerase is the major regulator 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. Previous studies on higher eukaryotes revealed that ubiquitination is one of the major post-translational modifications that affect the stability and activity of TERT component in telomerase holoenzyme. However, the role of ubiquitination in regulating RNA-protein interactions and telomerase activity remains unknown for any eukaryotic telomerase. Recently, we have characterized the interactome of the bloodstream form T. brucei parasites as part of the proposed activities in the parent proposal and found that T. brucei telomerase enzyme complex contains several ubiquitination - specific enzymes and factors. Additionally, new experimental data revealed that TERT protein is differentially ubiquitinated in the procyclic and bloodstream form developmental stages of T. brucei. Therefore, in this supplement proposal, PI intends to expand the original proposal’s objectives to elucidate the mechanisms of RNA-protein interactions of TERT and telomerase RNA during T.brucei development. Particularly, PI plans to map the ubiquitination sites on the TERT protein and determine their roles in T. brucei telomerase function. 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。这该多样性研究补充提案的总体目标是定义翻译后的作用端粒酶催化蛋白成分、RNA-蛋白相互作用中的 TERT 和端粒酶的修饰布氏锥虫的调控。 T. brucei 是一种早期分化的寄生原生生物，通过人类和昆虫的多种形态上不同的生命周期形式。在布氏锥虫中，端粒结构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的感染。端粒酶是布氏锥虫端粒合成的主要调节因子。两个高度保守端粒酶RNA结构域、RNA模板和eCR4/5独立地结合催化蛋白，端粒合成过程中端粒酶逆转录酶 (TERT) 是唯一必需的 RNA 元件用于催化活性端粒酶的体外重建。先前对高等真核生物的研究表明泛素化是影响TERT稳定性和活性的主要翻译后修饰之一端粒酶全酶的成分。然而，泛素化在调节 RNA 蛋白中的作用对于任何真核端粒酶来说，相互作用和端粒酶活性仍然未知。最近，我们有描述了布氏锥虫寄生虫血流的相互作用组，作为拟议活动的一部分父提案，发现T. brucei端粒酶复合物含有多个泛素化- 特定的酶和因子。此外，新的实验数据表明，TERT 蛋白具有差异性在布氏锥虫的发育阶段的前循环和血流中泛素化。因此，在这个补充提案中，PI打算扩展原始提案的目标，以阐明其机制 T.brucei 发育过程中 TERT 和端粒酶 RNA 的 RNA-蛋白质相互作用。特别是，PI 计划绘制 TERT 蛋白上的泛素化位点图并确定它们在 T. brucei 端粒酶功能中的作用。在综上所述，本研究将为 PI 定义核心组件的长期目标奠定基础临床中端粒酶的激活和相互作用对端粒长度稳态和基因组完整性的影响重要的原生生物。