Conserved vertebrate mir-451 provides a platform for Dicer-independent, Ago2-mediated microRNA biogenesis.
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 Slicer-deficient 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.
Yang
Jr-Shiuan
JS
Department of Developmental Biology, Sloan-Kettering Institute, New York, NY 10065, USA.
Maurin
Thomas
T
Robine
Nicolas
N
Rasmussen
Kasper D
KD
Jeffrey
Kate L
KL
Chandwani
Rohit
R
Papapetrou
Eirini P
EP
Sadelain
Michel
M
O'Carroll
Dónal
D
Lai
Eric C
EC
D000818:Animals; D060565:Argonaute Proteins; D001483:Base Sequence; D017124:Conserved Sequence; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004722:Endoribonucleases / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D034741:RNA, Small Interfering / Q000235:genetics; D043244:Ribonuclease III / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
C466422:AGO2 protein, human; C526735:Ago2 protein, mouse; D060565:Argonaute Proteins; D017931:DNA Primers; D015852:Eukaryotic Initiation Factor-2; C531201:MIRN451 microRNA, human; D035683:MicroRNAs; C550758:Mirn451 microRNA, mouse; D034741:RNA, Small Interfering; D004722:Endoribonucleases; C408087:DICER1 protein, human; C517918:DROSHA protein, human; C509781:Dicer1 protein, mouse; C513400:Drosha protein, mouse; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases