Structure-function analysis of a novel, maternally provided, multi-functional RNP complex in C. elegans

该项目提出了旨在表征的实验，无论是在体内还是在体外，一种新的蛋白质复合物在多个RNA相关的过程中的作用。该配合物命名为PETISCO，在C. elegans，由高度保守的和更多的C.线虫特异性蛋白质线虫特异性蛋白是PID-3和TOFU-6。PID-3具有MID-和RRM结构域，TOFU-6具有Tudor-和RRM结构域。高度保守的蛋白质是IFE-3（eIF 4 E的同源物）和ERH-2（线虫"残基增强子“的同源物之一）。后者是一种小蛋白，在S.粟酒PETISCO对于一类特定的小RNA（piRNA）的生物发生很重要，但也影响剪接前导RNA、SL 1和组蛋白mRNA。这些RNA种类以前没有机械地连接，因此PETISCO复合物代表了这些RNA种类之间的新颖且独特的界面。有趣的是，我们鉴定了两种蛋白质PID-1和TOST-1，它们相互排斥地结合PETISCO，并且似乎有助于定义PETISCO复合物的功能。这为我们提供了一个独特的手柄来研究这种多蛋白质复合物的不同方面。此外，我们已经阐明了不同PETISCO亚基之间的相互作用，为拟议的研究提供了一个现成的框架。该项目的目的是了解PETISCO的体外生物化学及其在体内的功能。重点将给予理解PETISCO功能的mRNA的反式剪接，和组蛋白mRNA的稳态。将使用的技术将从重组蛋白质技术和结构生物学到基因组编辑和C.优雅

This project presents experiments aimed at characterizing, both in vivo as well as in vitro, a novel protein complex with roles in multiple RNA-related processes. This complex, named PETISCO and identified in C. elegans, is composed of both highly conserved, and more C. elegans specific proteins. Nematode-specific proteins are PID-3 and TOFU-6. PID-3 has a MID- and an RRM domain, and TOFU-6 has a Tudor- and an RRM domain. Strongly conserved proteins are IFE-3, a homolog of eIF4E and ERH-2, one of the nematode homologs of 'enhancer of rudimentary'. The latter is a small protein, with known roles, even though mechanistically unresolved, in mRNA homeostasis in S. pombe. PETISCO is important for the biogenesis of a specific class of small RNAs (piRNAs), but also affects the splice-leader RNA, SL1, and histone mRNA. These RNA species have not mechanistically been linked before, and as such the PETISCO complex represents a novel and unique interface between these RNA species. Interestingly, we identified two proteins, PID-1 and TOST-1, that bind PETISCO mutually exclusively, and that appear to help define the function of the PETISCO complex. This provides us with a unique handle to study different aspects of this multi-protein complex. Furthermore, we already elucidated the interactions between the different PETISCO subunits, providing a ready-to-go framework for the proposed studies. The aim of the project is to understand PETISCO biochemistry in vitro and its function in vivo. Emphasis will be given to understanding PETISCO function in relation to trans-splicing of mRNAs, and to histone mRNA homeostasis. Techniques that will be used will range from recombinant protein technology and structural biology to genome editing and phenotypic analyses in C. elegans.