Argonaute-bound small RNAs from promoter-proximal RNA Polymerase II

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