Abstract
RNA interference (RNAi) is an effective mechanism for inhibiting gene expression at the post-transcriptional level. Expression of a messenger RNA (mRNA) can be inhibited by a ∼22-nucleotide (nt) small interfering (si)RNA with the corresponding reverse complementary sequence. Typically, a duplex of siRNA, composed of the desired siRNA and a passenger strand, is processed from a short hairpin RNA (shRNA) precursor by Dicer. Subsequently, one strand of the siRNA duplex is associated with Argonaute (Ago) protein for RNAi. Although RNAi is widely used, the off-target effect induced by the passenger strand remains a potential problem. Here, based on current understanding of endogenous precursor microRNA (pre-miRNA) hairpins, called Ago-shRNA and m7G-capped pre-miRNA, we discuss the principles of shRNA designs that produce a single siRNA from one strand of the hairpin.
Highlights
Gene expression can be regulated at the post-transcriptional level in a number of ways
Pol III initiates transcription at a precise position starting with a purine and ends with two additional Us to produce short hairpin RNA (shRNA) mimicking the structure of a pre-miRNA, which has a 2-nt overhang at the 3’ end
Pre-miR-451 is too shRNA for Single siRNA Expression short to be recognized by Dicer and is directly bound by Ago2
Summary
Gene expression can be regulated at the post-transcriptional level in a number of ways. Pol III initiates transcription at a precise position starting with a purine and ends with two additional Us to produce shRNA mimicking the structure of a pre-miRNA, which has a 2-nt overhang at the 3’ end. The shRNA hairpin exits the nucleus with the assistance of XPO5, and can be further processed by Dicer into ∼22 bp siRNA duplexes following the 5’-end, 3’-end and the loop- counting rules similar to a pre-miRNA (Park et al, 2011; Gu et al, 2012; Tian et al, 2014).
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