FUNCTIONAL PROTEOMICS REVEALS THE BIOCHEMICAL NICHE OF C ELEGANS DCR-1 IN MULT

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在植物、动物和真菌中，与RNaseIII相关的酶家族中的DICER家族成员处理双链RNA(DsRNA)来启动小RNA介导的基因沉默机制。为了了解线虫DICER，DCR-1是如何在多种不同的沉默机制中发挥作用的，我们使用了基于质谱学的蛋白质组学方法来鉴定DCR-1相互作用的蛋白质。然后，我们产生了相应基因的缺失等位基因，并对其进行了表征。这些相互作用元件是产生三种小RNA所必需的，包括(1)来自外源dsRNA触发器(exo-siRNAs)的小干扰RNAs(SiRNAs)；(2)来自内源触发器(Endo-siRNAs)的siRNAs；(3)发育调节microRNAs(MiRNAs)。一个相互作用子，保守的RNA-磷酸酶同系物PIR-1，是处理可能扩增的DCR-1底物所必需的。产生Endo-siRNA所需的相互作用因子包括ERI-1和RRF-3，它们的功能丧失会增强RNAi。我们的发现首次让人们得以一窥DICER复杂的生化生态位，并表明DCR-1介导的小RNA途径之间存在竞争。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In plants, animals, and fungi, members of the Dicer family of RNase III-related enzymes process double-stranded RNA (dsRNA) to initiate small-RNA-mediated gene-silencing mechanisms. To learn how C. elegans Dicer, DCR-1, functions in multiple distinct silencing mechanisms, we used a mass-spectrometry-based proteomics approach to identify DCR-1-interacting proteins. We then generated and characterized deletion alleles for the corresponding genes. The interactors are required for production of three species of small RNA, including (1) small interfering RNAs (siRNAs), derived from exogenous dsRNA triggers (exo-siRNAs); (2) siRNAs derived from endogenous triggers (endo-siRNAs); and (3) developmental regulatory microRNAs (miRNAs). One interactor, the conserved RNA-phosphatase homolog PIR-1, is required for the processing of a putative amplified DCR-1 substrate. Interactors required for endo-siRNA production include ERI-1 and RRF-3, whose loss of function enhances RNAi. Our findings provide a first glimpse at the complex biochemical niche of Dicer and suggest that competition exists between DCR-1-mediated small-RNA pathways.