Decoding RNA-Protein Interactions in Trypanosoma Telomerase

项目摘要 端粒酶是一种独特的核糖核蛋白酶，它不断地增加端粒重复序列，从其 完整的RNA组分，位于线形染色体的末端，以防止真核生物中基因组的不稳定性。这 一项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 布氏锥虫是一种早期发散的寄生原生原生动物，通过多个 人类和昆虫在形态上截然不同的生命周期形式。在布氏锥虫中，端粒结构扮演着一种 在调节抗原变异方面发挥重要作用，使寄生虫能够建立长期感染。 特别是，极短的端粒可能危及端粒的完整性，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 布氏毛滴虫的复制可为控制寄生虫的增殖提供重要线索。端粒酶是主要的 布氏毛滴虫端粒合成的机制。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，是催化体外重组所需的唯一RNA元件 端粒酶活性。然而，布氏毛滴虫端粒酶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，提示了新的模式 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域的多样性导致RNA-蛋白质相互作用的差异 构象变化，导致特定物种的端粒酶组装和活性。我们最近的RNA 布鲁氏毛滴虫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。要了解特定阶段的结构重组是如何 和RNA-蛋白质的相互作用控制着布鲁氏锥虫的端粒酶调控，在提案的目标1中，我们将 布氏毛滴虫上述两个结构域中RNA-蛋白质相互作用的分子需求 端粒酶。这项提案的目标2将通过解剖探索端粒酶调节的额外要求 端粒酶RNP组装和活性所需的RNA特异性因子。总而言之，这是 研究将为PI定义端粒酶激活的核心成分的长期目标奠定基础 以及在临床上重要的原生生物中端粒长度、动态平衡和基因组完整性的相互作用。

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.