Intertwined pathways for Argonaute-mediated microRNA biogenesis in Drosophila.
Although Dicer is essential for general microRNA (miRNA) biogenesis, vertebrate mir-451 is Dicer independent. Instead, its short pre-miRNA hairpin is 'sliced' by Ago2, then 3'-resected into mature miRNAs. Here, we show that Drosophila cells and animals generate functional small RNAs from mir-451-type precursors. However, their bulk maturation arrests as Ago-cleaved pre-miRNAs, which mostly associate with the RNAi effector AGO2. Routing of pre-mir-451 hairpins to the miRNA effector AGO1 was inhibited by Dicer-1 and its partner Loqs. Loss of these miRNA factors promoted association of pre-mir-451 with AGO1, which sliced them and permitted maturation into ∼ 23-26 nt products. The difference was due to the 3' modification of single-stranded species in AGO2 by Hen1 methyltransferase, whose depletion permitted 3' trimming of Ago-cleaved pre-miRNAs in AGO2. Surprisingly, Nibbler, a 3'-5' exoribonuclease that trims 'long' mature miRNAs in AGO1, antagonized miR-451 processing. We used an in vitro reconstitution assay to identify a soluble, EDTA-sensitive activity that resects sliced pre-miRNAs in AGO1 complexes. Finally, we use deep sequencing to show that depletion of dicer-1 increases the diversity of small RNAs in AGO1, including some candidate mir-451-like loci. Altogether, we document unexpected aspects of miRNA biogenesis and Ago sorting, and provide insights into maturation of Argonaute-cleaved miRNA substrates.
Department of Developmental Biology, Sloan-Kettering Institute, 1275 York Ave, Box 252, New York, NY 10065, USA and Molecular Biology Program, Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10065, USA.
Jr-Shiuan
JS
Yang
Peter
P
Smibert
Jakub O
JO
Westholm
David
D
Jee
Thomas
T
Maurin
Eric C
EC
Lai
D000818:Animals; D060565:Argonaute Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004330:Drosophila / Q000235:genetics* / Q000378:metabolism; D029721:Drosophila Proteins / Q000378:metabolism*; D005095:Exoribonucleases / Q000378:metabolism; D008780:Methyltransferases / Q000378:metabolism; D035683:MicroRNAs / Q000378:metabolism*; D020365:RNA Helicases / Q000378:metabolism; D034622:RNA Interference; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D043244:Ribonuclease III / Q000378:metabolism
C489654:AGO1 protein, Drosophila; C433807:AGO2 protein, Drosophila; D060565:Argonaute Proteins; D029721:Drosophila Proteins; C503213:Loqs protein, Drosophila; D035683:MicroRNAs; D012322:RNA Precursors; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C521948:Hen1 protein, Drosophila; D008780:Methyltransferases; C515033:Dcr-1 protein, Drosophila; D005095:Exoribonucleases; C570892:Nbr protein, Drosophila; D043244:Ribonuclease III; D020365:RNA Helicases