Conserved vertebrate mir-451 provides a platform for Dicer-independent, Ago2-mediated microRNA biogenesis

Abstract

Canonical animal microRNAs (miRNAs) are generated by sequential cleavage of precursor substrates by the Drosha and Dicer RNase III enzymes. Several variant pathways exploit other RNA metabolic activities to generate functional miRNAs. However, all of these pathways culminate in Dicer cleavage, suggesting that this is a unifying feature of miRNA biogenesis. Here, we show that maturation of miR-451, a functional miRNA that is perfectly conserved among vertebrates, is independent of Dicer. Instead, structure-function and knockdown studies indicate that Drosha generates a short pre-mir-451 hairpin that is directly cleaved by Ago2 and followed by resection of its 3′ terminus. We provide stringent evidence for this model by showing that Dicer knockout cells can generate mature miR-451 but not other miRNAs, whereas Ago2 knockout cells reconstituted with wild-type Ago2, but not Slicerdeficient Ago2, can process miR-451. Finally, we show that the mir-451 backbone is amenable to reprogramming, permitting vector-driven expression of diverse functional miRNAs in the absence of Dicer. Beyond the demonstration of an alternative strategy to direct gene silencing, these observations open the way for transgenic rescue of Dicer conditional knockouts.Slicer | gene suppression | miRNA reprogramming

The Potential Role of miR-451 in Cancer Diagnosis, Prognosis, and Therapy

“…1C). Interestingly, Cifuentes and colleagues observed that Ago2 protected the approximately 30-nt slicercleaved intermediate from RNase I in vitro, resulting in a approximately 20-to 26-nt 3 0 -end trimmed product, similar to the observations by Yang and colleagues (5,7,31,32). Recently, Yang and colleagues show that the degree of G:C content in the resected region governs the maturation efficiency of 3 0 resection and resultant regulatory activity, such that a relatively low G:C content improves processing (31).…”

“…The 42-nt Drosha-cleaved miR-451 hairpin with a 17-nt stem does not possess a sufficiently long duplex to be a Dicer substrate (>19 nt), suggesting that its maturation might bypass a requirement for Dicer (27). Recently, 3 studies found by coincidence that the maturation of miR-451 seemed refractory to loss-of-function mutations in Dicer (5,7,10). miR-451 accumulated effectively in embryonic stem cells carrying a conditional knockout of Dicer (10), in mouse embryonic fibroblasts (MEF) stably deleted for Dicer (5) and in MZdicer mutant zebrafish (7).…”

“…1B; ref. 5). Broad miRNA expression profiling analyses have recurrently identified miR-144 and miR-451 for their high erythropoietic cell-restricted expression in several species, including zebrafish (4, 6, 7), mice (8)(9)(10), and human (5,11,12).…”

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

“…For miR-451, oligouridylation at the 3 ′ end and subsequent 3 ′ trimming occurs to reach the mature miRNA length Cifuentes et al 2010;Yang et al 2010), and the poly(A)-specific endonuclease PARN was recently implicated (Yoda et al 2013). Surprisingly, trimming was found not to be essential for subsequent target mRNA silencing in vitro and in vivo (Yoda et al 2013).…”

“…However, there is a growing body of evidence for the existence of noncanonical or alternative processing routes for natural miRNAs and manmade shRNAs. 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).…”