Abstract
Recent evidence indicates the presence of alternative pathways for microRNA (miRNA) and short hairpin (shRNA) processing. Specifically, some of these molecules are refractory to Dicer-mediated processing, which allows alternative processing routes via the Ago2 endonuclease. The resulting RNA molecules differ in size and sequence and will thus trigger the silencing of different target RNAs. It is, therefore, important to understand these processing routes in mechanistic detail such that one can design exclusive RNA reagents for a specific processing route. The exact sh/miRNA properties that determine this routing toward Dicer or Ago2 are incompletely understood. The size of the base-paired stem seems an important determinant, but other RNA elements may contribute as well. In this study, we document the importance of a weak G-U or U-G base pair at the top of the hairpin stem.
Highlights
Small noncoding microRNAs regulate cellular gene expression at the post-transcriptional level (Brummelkamp et al 2002; Bartel 2009; Carthew and Sontheimer 2009). These miRNAs are usually processed by the nuclear Drosha endonuclease, transported to the cytoplasm by Exportin-5, further processed by the Dicer endonuclease, and subsequently associate with an Argonaute (Ago) protein in the RNA-induced silencing complex (RISC) to induce degradation of complementary target RNAs (Filipowicz et al 2008; Siomi and Siomi 2009). This RNA interference (RNAi) mechanism can be triggered by man-made gene constructs that express short hairpin RNA molecules in the nucleus that enter the RNAi pathway at the Dicer processing step in the cytoplasm, followed by silencing of the complementary mRNA target
We tested the hypothesis that a weak G-U base pair at the top of the short hairpin RNA (shRNA) stem is advantageous for using the noncanonical Ago2-processing route instead of regular Dicer-processing
This effect was observed for several mutant shRNA sets in reporter assays and Northern blot analyses that discriminate between these two pathways
Summary
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). The larger hairpins (21–23 bp) maintained quite a lot of activity on the Luc-anti-sense reporter, but this is, in part, due to passenger strand activity of the regular shRNA-route These combined results provide the first evidence that a weak top base pair may, favor AgoshRNA processing over the regular shRNA-route. Probing with a small oligonucleotide for the 3′ side of the molecule should detect the regular Dicer-processed guide strand of ∼21 bp (Fig. 4, top panel) This fragment was abundantly present but only for shRNAs of 20 or more base pairs, which nicely correlates with the activity data. Compared to shRT5, the mutant constructs showed similar activity on the Luc-anti-sense reporter Please realize that this reporter will detect the activity of both the AgoshRNA strand and the passenger strand of regularly Dicer-processed shRNAs (gray and white arrows, respectively). Maintained full AgoshRNA activity of 19/5 on the Luc-antisense reporters in these mutant Dicer cells (data not shown)
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