Biogenesis and Function of Guide RNAs

描述(申请人提供)：锥虫是一种寄生原虫，对发展中国家的健康问题负责，包括那些在美国有大量存在的国家。除了是一种重要的病原体外，布鲁氏锥虫还提供了一个扩展的分子生物学图景，超出了标准模式生物集的范围。事实上，在这种寄生虫中已经发现了许多独特的生物学过程。特别是，它的巨大线粒体包裹着一种不寻常的DNA结构，称为动泡体，以及非传统的RNA处理途径。动植体携带一个编码蛋白质的上环基因组，连接到编码大量引导RNA(GRNA)的小环上。许多核编码因子通过核溶解处理、U-插入/缺失RNA编辑和32多聚腺苷作用，调节大环和小环转录本之间的相互作用，从而产生功能性的mRNAs。编辑反应由RNA编辑核心复合体RECC(20S编辑小体)催化，而酶级联反应的每一步都由gRNA指导。尽管对RECC的研究已经显示出令人印象深刻的进展，但直到最近，人们对gRNA的生物发生、稳定性、编辑过程的招募以及编辑后的代谢命运知之甚少。我们发现，RNA编辑TUTase 1(RET1)参与了gRNA前体的核溶解过程，成熟的gRNA通过与gRNA结合复合体亚基1和2(GRBC1/2)结合而稳定。在初步研究中，我们鉴定了与GRBC1/2相关的蛋白质，包括NUDIX水解酶、DExD/H RNA解旋酶、RNA结合蛋白和缺乏可识别结构域的多肽。GRBC的整体复杂性可能超过RECC，RECC由约20种蛋白质组成。本建议侧重于GRBC的蛋白质组成、gRNA结合和对编辑过程的承诺机制以及编辑后的gRNA置换。我们假设RECC和GRBC相互作用形成一个RNA编辑全酶，称为40S编辑体，并建议：1)定义核心蛋白质组分和那些参与GRBC内RNA介导的联系的组分；2)确定gRNA与GRBC1/2结合的机制以及GRBC-RECC的相互作用；以及3)研究编辑后gRNA置换的分子基础。

DESCRIPTION (provided by applicant): Trypanosomes are parasitic protozoa responsible for health problems in developing countries, including those with a substantial U.S. presence. Along with being an important pathogen, Trypanosoma brucei offers an expanded molecular biology landscape that extends beyond that of the standard set of model organisms. Indeed, many unique biological processes have been discovered in this parasite. In particular, its giant mitochondrion encloses an unusual DNA structure, referred to as the kinetoplast, and unconventional RNA processing pathways. The kinetoplast carries a protein-coding maxicircle genome catenated to minicircles which encode a vast array of guide RNAs (gRNAs). A multitude of nuclear-encoded factors mediate the interactions between maxi- and minicircle transcripts to create functional mRNAs via nucleolytic processing, U- insertion/deletion RNA editing, and 32 polyadenylation. Editing reactions are catalyzed by the RNA editing core complex, RECC (20S editosome), while each step of the enzymatic cascade is directed by gRNAs. Although studies of RECC have shown impressive progress, until recently little was known about gRNA biogenesis, stabilization, recruitment to the editing process, and post-editing metabolic fate. We discovered that RNA editing TUTase 1 (RET1) is involved in the nucleolytic processing of gRNA precursors and that mature gRNAs are stabilized via binding to gRNA binding complex subunits 1 and 2 (GRBC1/2). In preliminary studies we identified GRBC1/2-associated proteins, including NUDIX hydrolases, DExD/H RNA helicases, RNA binding proteins, and polypeptides lacking identifiable domains. The overall complexity of GRBC likely exceeds that of the RECC, which consists of ~20 proteins. This proposal focuses on GRBC protein composition, mechanisms of gRNA binding and commitment to the editing process, and post-editing gRNA displacement. We hypothesize that interacting RECC and GRBC form an RNA editing holoenzyme, termed the 40S editosome, and propose to: 1) define core protein components and those involved in RNA-mediated contacts within GRBC; 2) determine the mechanisms of gRNA binding to GRBC1/2 as well as GRBC-RECC interaction; and 3) investigate the molecular basis of post-editing gRNA displacement.