Collaborative Research: RNA Processing in Trypanosome Mitochondria

这个项目将研究古代单细胞寄生虫中一种独特的基因表达机制，称为锥虫。在大多数生物中，RNA是从DNA复制而来的，并在不改变密码的情况下指导蛋白质的合成。然而，在锥虫线粒体(细胞的‘动力库’)中，RNA通过添加或移除特定核苷酸而被广泛编辑。在其复杂的生命周期中，锥虫在两种截然不同的宿主之间交替：人类(和其他动物)和昆虫，这两种宿主是传播媒介。寄生虫在人类和昆虫身上面临的环境截然不同，需要快速和大规模的代谢和生理变化，包括RNA编辑。在寄生虫发育过程中如何精确调控RNA编辑机制这一关键问题仍未得到回答，也是本研究的重点。该项目将通过为博士后、研究生、本科生和高中生提供跨学科培训机会，产生广泛的教育影响，重点关注在STEM学科中传统上代表性较低的群体。该项目重点研究布氏锥虫mRNA编辑调控和控制编辑小体编辑保真度的REH2 C复合体的关键作用。这项研究可以为一个几十年来一直是个谜的问题提供新的线索：在布氏锥虫的整个生命周期中，mRNA编辑是如何受到差异控制的？目的1测试部分编辑控制(PEC)模型，该模型利用部分编辑来不同地抑制布氏毛滴虫两种发育形式的mRNA成熟。为此，将使用最近开发的用于编辑RNA的RNA-SEQ/生物信息学分析的流水线。目标2将解决编辑中底物专一性的来源，特别是确定一个罕见的Deah-box解旋酶辅助因子及其单个锌指在机制中的作用。目标3将通过利用遗传学、生化(邻近标记)和化学(交联-质谱学)方法阐明编辑小体的一般组织，来研究编辑成分与REH2 C复合体重塑的动态相互作用。总体而言，这些研究将揭示寄生虫两个生命周期阶段编辑规则的新方面，包括编辑保真度、底物专一性决定因素和编辑体相互作用。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This project will study a distinctive mechanism of gene expression in ancient single-celled parasites called trypanosomes. In most organisms, RNA is copied from DNA and directs protein synthesis without changes in the code. However, in trypanosomal mitochondria (the ‘powerhouse’ of cells), RNA is edited extensively by addition or removal of specific nucleotides. In their complex life cycle, trypanosomes alternate between two very different hosts: humans (and other animals) and insects, which are the vectors of tranmission. The dramatically different environments that the parasites face in humans and insects demand rapid and large-scale metabolic and physiological changes, including in RNA editing. The crucial question of how the RNA editing mechanism is precisely regulated during parasite development remains unanswered and is the focus of this research. The project will have broad educational impact by providing interdisciplinary training opportunities for postdoctoral, graduate, undergraduate, and high school students, with a focus on groups traditionally underrepresented in the STEM disciplines.This project focuses on Trypanosoma brucei mRNA editing regulation and the key role of the REH2C complex that controls editing fidelity in editosomes. The research can shed new light on a question that has remained a mystery for decades: how is mRNA editing differentially controlled across the T. brucei life-cycle? Objective 1 will test the “partial-editing control” (PEC) model of exploiting partial editing to differentially inhibit mRNA maturation in two developmental forms of T. brucei. A recently developed pipeline for RNA-seq/bioinformatic analysis of edited RNAs will be employed for this purpose. Objective 2 will address the source of substrate specificity in editing, specifically to define the role of a rare DEAH-box helicase cofactor and its individual zinc fingers in the mechanism. Objective 3 will examine dynamic interactions of editing components and remodeling by the REH2C complex by elucidating the general organization of editosomes using genetic, biochemical (proximity labeling), and chemical (crosslinking-mass spectrometry) approaches. Overall, these studies will reveal new aspects of editing regulation in two life-cycle stages of the parasite, including editing fidelity, substrate specificity determinants, and editosome interactions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.