Biogenesis and Function of Guide RNAs

描述（由申请人提供）：锥虫是一种寄生原生动物，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。作为一种重要的病原体，布鲁氏锥虫提供了一个扩展的分子生物学景观，超出了标准模式生物的范围。事实上，在这种寄生虫中发现了许多独特的生物过程。特别是，它巨大的线粒体包裹着一种不寻常的DNA结构，称为着丝体，以及非常规的RNA加工途径。着丝质体携带一个编码蛋白质的大环基因组，它被连成小环，编码大量的引导rna （gRNAs）。大量的核编码因子介导大环和小环转录本之间的相互作用，通过核分解加工、U-插入/删除RNA编辑和32多聚腺苷化产生功能性mrna。编辑反应由RNA编辑核心复合体RECC （20S编辑体）催化，而酶级联的每一步都由grna指导。尽管对RECC的研究取得了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、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gRNA前体的核分解加工，成熟的gRNA通过与gRNA结合复合物亚基1和2 （GRBC1/2）结合而稳定。在初步研究中，我们确定了grbc1 /2相关蛋白，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的总体复杂性可能超过RECC，后者由约20种蛋白质组成。本提案主要关注GRBC蛋白组成、gRNA结合机制和对编辑过程的承诺，以及编辑后gRNA的位移。我们假设相互作用的RECC和GRBC形成了一种称为40S编辑体的RNA编辑全酶，并提出：1)定义GRBC中核心蛋白成分和参与RNA介导接触的蛋白成分；2)确定gRNA与GRBC1/2结合及GRBC-RECC相互作用的机制；3)研究编辑后gRNA位移的分子基础。

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.