Dicer-independent processing of short hairpin RNAs

Abstract

Short hairpin RNAs (shRNAs) are widely used to induce RNA interference (RNAi). We tested a variety of shRNAs that differed in stem length and terminal loop size and revealed strikingly different RNAi activities and shRNA-processing patterns. Interestingly, we identified a specific shRNA design that uses an alternative Dicer-independent processing pathway. Detailed analyses indicated that a short shRNA stem length is critical for avoiding Dicer processing and activation of the alternative processing route, in which the shRNA is incorporated into RISC and processed by the AGO2-mediated slicer activity. Such alternatively processed shRNAs (AgoshRNAs) yield only a single RNA strand that effectively induces RNAi, whereas conventional shRNA processing results in an siRNA duplex of which both strands can trigger RNAi. Both the processing and subsequent RNAi activity of these AgoshRNAs are thus mediated by the RISC-component AGO2. These results have important implications for the future design of more specific RNAi therapeutics.

Probing the shRNA characteristics that hinder Dicer recognition and consequently allow Ago-mediated processing and AgoshRNA activity

“…that attacks the shRNA loop (Dallas et al 2012), but deep sequencing did not point to such a possibility (Liu et al 2013).…”

“…Whereas Dicer is critical for activation of the majority of these miRNA and shRNA molecules, notable exceptions were reported for a special set of miRNAs Cifuentes et al 2010;Miyoshi et al 2010;Yang et al 2010;Yang and Lai 2011;Havens et al 2012). More recently, Dicer-independent shRNAs have been described as well (Ge et al 2010;Dallas et al 2012;Liu et al 2013). We called these molecules AgoshRNAs because Ago2 is involved in their processing (Liu et al 2013).…”

“…More recently, Dicer-independent shRNAs have been described as well (Ge et al 2010;Dallas et al 2012;Liu et al 2013). We called these molecules AgoshRNAs because Ago2 is involved in their processing (Liu et al 2013).…”

“…A detailed mutational analysis indicated that the length of the base-paired stem is the major determinant for shRNA activity via the regular Dicer route vs. AgoshRNA activity via the noncanonical Ago2 route (Liu et al 2013). shRNAs of 19 bp or less lose the ability to be processed by Dicer, which opens up the alternative Ago2-processing route for AgoshRNAs.…”

“…shRNAs of 19 bp or less lose the ability to be processed by Dicer, which opens up the alternative Ago2-processing route for AgoshRNAs. AgoshRNAs remain active down to 17 bp, but shorter hairpins lose all activity (Liu et al 2013). However, other sequence and/or structure elements may also influence the routing of shRNAs.…”