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

本项目提出了旨在体内和体外表征的实验，一种在多个rna相关过程中起作用的新型蛋白质复合物。这个复合物被命名为PETISCO，在秀丽隐杆线虫中发现，由高度保守和更多秀丽隐杆线虫特异性蛋白质组成。线虫特异性蛋白是PID-3和TOFU-6。PID-3具有MID-和RRM域，而TOFU-6具有Tudor-和RRM域。强保守蛋白是eIF4E和ERH-2的同源物IFE-3， ERH-2是“初级增强子”的线虫同源物之一。后者是一种小蛋白，在S. pombe的mRNA稳态中具有已知的作用，尽管机制尚未确定。PETISCO对一类特定小RNA （pirna）的生物发生很重要，但也影响剪接先导RNA、SL1和组蛋白mRNA。这些RNA物种之前没有机械地连接，因此PETISCO复合物代表了这些RNA物种之间新颖而独特的界面。有趣的是，我们发现了两个蛋白，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。这为我们研究这种多蛋白复合物的不同方面提供了一个独特的方法。此外，我们已经阐明了不同的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体外生物化学及其在体内的功能。重点将放在理解与mRNA反式剪接和组蛋白mRNA稳态有关的PETISCO功能。将使用的技术范围从重组蛋白技术和结构生物学到秀丽隐杆线虫的基因组编辑和表型分析。

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.