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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。在植物、动物和真菌中，RNase III相关酶Dicer家族的成员加工双链RNA（dsRNA）以启动小RNA介导的基因沉默机制。要了解C.线虫Dicer，DCR-1，在多种不同的沉默机制中发挥作用，我们使用基于质谱的蛋白质组学方法来鉴定DCR-1相互作用蛋白。然后，我们生成并表征相应基因的缺失等位基因。所述相互作用物是产生三种小RNA所必需的，包括（1）衍生自外源dsRNA触发物（exo-siRNA）的小干扰RNA（siRNA）;（2）衍生自内源触发物（endo-siRNA）的siRNA;和（3）发育调节微RNA（miRNA）。一个相互作用，保守的RNA-磷酸酶同源物PIR-1，需要处理一个假定的扩增的DCR-1底物。产生内siRNA所需的相互作用物包括ERI-1和RRF-3，其功能丧失增强RNAi。我们的研究结果提供了第一个一瞥在复杂的生物化学生态位的切丁，并表明竞争存在于DCR-1介导的小RNA途径。

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.