Dicer-independent, Ago2-mediated microRNA biogenesis in vertebrates

Abstract

A canonical biogenesis pathway involving sequential cleavage by the Drosha and Dicer RNAse III enzymes governs the maturation of most animal microRNAs. However, there exist a variety of alternative miRNA biogenesis pathways, most of which bypass Drosha processing. Recently, three groups described for the first time a vertebrate microRNA pathway that bypasses Dicer cleavage. This mechanism was characterized with respect to the highly conserved vertebrate gene mir-451, for which Drosha processing yields a short (42 nucleotide) hairpin that is directly loaded into Ago2, the sole vertebrate "Slicer" Argonaute. Ago2-mediated cleavage of this hairpin yields a 30 nucleotide intermediate, whose 3' end is resected to generate the dominantly cloned ~23 nucleotide mature miR-451. Knowledge of this pathway provides an unprecedented tool with which to express microRNAs and small interfering RNAs in Dicer mutant cells. More generally, the mir-451 backbone constitutes a new platform for gene silencing that complements existing shRNA technology.

Figure 1

(A) Canonical miRNA biogenesis. Primary miRNA (pri-miRNA) transcripts are cleaved in the nucleus by a complex of the Drosha RNAse III enzyme and its partner DGCR8 to yield a ∼55–70 nt pre-miRNA hairpin. Following its translocation to the cytoplasm via Exportin-5, the pre-miRNA hairpin is cleaved on its loop end by a complex of the Dicer RNAse III enzyme and its partner TRBP/PACT. The resultant ∼22 nt miRNA/miRNA* duplex is loaded into any of the 4 vertebrate Argonaute (AGO) proteins and one strand is released leaving behind the mature miRNA. (B) miR-451 biogenesis. mir-451 resides on a primary transcript operon with mir-144, which matures via the canonical miRNA pathway. Drosha/DGCR8 cleavage generates a 42 nt pre-mir-451 hairpin, which resembles an Exportin-5 substrate (although this has not been directly demonstrated, thus the “?”). The pre-mir-451 hairpin is directly loaded into AGO proteins, but loading into non-slicing AGO proteins is abortive and these cannot mature mir-451 further. Loading into AGO2, the sole vertebrate “Slicer” capable of cleaving target strands, generates a 30 nt “Ago2-cleaved (ac)-pre-mir-451 hairpin.” This is then subject to a trimming reaction, which may occur in conjunction with a tailing reaction, to yield the dominant 23 nt mature miR-451.