Argonaute-bound small RNAs from promoter-proximal RNA Polymerase II. Mus musculus

Argonaute (Ago) proteins mediate post-transcriptional gene repression by binding guide microRNAs (miRNAs) to regulate targeted RNAs. To confidently assess Agobound small RNAs, we adapted a mouse embryonic stem cell system to express a single inducible epitope-tagged Ago protein. Here, we report the small RNA profile of Agodeficient cells and determine Ago-dependent stability is a common feature of mammalian miRNAs. Considering both in vivo Ago-dependence for stability and Ago2 binding as defined by immunopurification, we have identified a novel class of non-canonical miRNAs derived from protein-coding gene promoters, which we name transcriptional start site miRNAs (TSS-miRNAs). A subset of promoter-proximal RNA polymerase II complexes produce hairpin RNAs that are processed in a DGCR8/Drosha-independent, but Dicer-dependent manner. TSS-miRNA activity is detectable endogenously, upon transfection of a mimic or by mRNA overexpression. Finally, we present evidence of differential expression and conservation in humans, suggesting important roles in gene regulation. Overall design: Examination of Ago immunoprecipitations and mESC without Ago proteins

Argonaute（Ago）蛋白通过结合向导microRNA（miRNA）调控靶RNA，介导转录后基因沉默。为精准评估Ago结合的小RNA，我们构建了可表达单一可诱导表位标记Ago蛋白的小鼠胚胎干细胞系统。本研究报道了Ago缺陷细胞的小RNA表达谱，并证实Ago依赖的稳定性是哺乳动物miRNA的普遍特征。综合体内Ago依赖的稳定性特性与免疫纯化鉴定的Ago2结合数据，我们鉴定出一类源自蛋白编码基因启动子的新型非经典miRNA，将其命名为转录起始位点miRNA（TSS-miRNA）。一类启动子近端的RNA聚合酶II（RNA polymerase II）复合物可生成发夹RNA，该类RNA的加工过程不依赖DGCR8/Drosha，但依赖Dicer。TSS-miRNA的活性可在内源条件下被检测到，也可通过转染模拟物或mRNA过表达得以验证。最后，我们提供了其在人类中存在差异表达与保守性的证据，提示其在基因调控中发挥重要功能。实验整体设计：对Ago免疫沉淀样本与不含Ago蛋白的小鼠胚胎干细胞进行检测。