Argonaute 2‐loaded promoter‐targeted double stranded RNA (Ago2‐ptRNA) mediates gene activation by interacting with the core transcriptional machinery

Abstract

Promoter‐targeted double‐stranded RNA (ptRNA) has been shown to mediate potent activation of gene expression in a phenomenon known as RNA activation (RNAa). Previous studies revealed requirement of RNAa on Argonaute 2 (Ago2) protein as well as RNAa association with epigenetic changes at the target promoter. However, its precise molecular nature remains elusive.Using human p21 promoter as a model system, we further characterized RNAa mechanism. ptRNA designed to target p21 promoter is loaded by Ago2 protein, which uses one of the RNA strands to guide it binding to promoter DNA. ptRNA‐Ago2 binding to the promoter target site activate p21 gene expression on transcriptional level. Our biochemical studies revealed the identity of the RNAa effector complex, which contains ptRNA‐loaded Ago2 protein and at least two additional protein components: RNA helicase A (RHA) and RNA polymerase II (RNAP II)‐associated factor CTR9, both capable of recruiting histone modifying enzymes to facilitate transcription initiation and elongation. We show evidence that RNAa effector complex physically associates with RNAPII to facilitate re‐initiation of transcription.Together, our results provide important new insights into RNAa mechanism and allow us to build a working model for RNAa.The research was supported by grants from NIH (1R01GM090293–0109 to L.C.L.) and Department of Defense (W81XWH‐08–1‐0260 to L.C.L.)