Analysis of AgoshRNA maturation and loading into Ago2
by Alex Harwig, Zita Kruize, Zhenhuang Yang, Tobias Restle, Ben BerkhoutThe RNA interference (RNAi) pathway was recently expanded by the discovery of multiple alternative pathways for processing of natural microRNA (miRNA) and man-made short hairpin RNA (shRNA) molecules. One non-canonical pathway bypasses Dicer cleavage and requires instead processing by Argonaute2 (Ago2), which also executes the subsequent silencing step. We named these molecules AgoshRNA, which generate only a single active RNA strand and thus avoid off-target effects that can be induced by the passenger strand of a regular shRNA. Previously, we characterized AgoshRNA processing by deep sequencing and demonstrated that—after Ago2 cleavage—AgoshRNAs acquire a short 3’ tail of 1–3 A-nucleotides and are subsequently trimmed, likely by the poly(A)-specific ribonuclease (PARN). As a result, the mature single-stranded AgoshRNA may dock more stably into Ago2. Here we set out to analyze the activity of different synthetic AgoshRNA processing intermediates. Ago2 was found to bind preferentially to partially single-stranded AgoshRNA in vitro. In contrast, only the double-stranded AgoshRNA precursor associated with Ago2 in cells, correlating with efficient intracellular processing and reporter knockdown activity. These results suggest the presence of a cellular co-factor involved in AgoshRNA loading into Ago2 in vivo. We also demonstrate specific AgoshRNA loading in Ago2, but not Ago1/3/4, thus further reducing unwanted side effects.
Publisher URL: http://journals.plos.org/plosone/article
Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.
Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.