title: Functional parameters of Dicer-independent microRNA biogenesis.
abstract: Until recently, a Dicer-class RNase III enzyme was believed to be essential for microRNA (miRNA) biogenesis in all animals. The conserved vertebrate locus mir-451 defies this expectation and instead matures by direct cleavage of its pre-miRNA hairpin via the Slicer activity of Argonaute2 (Ago2). In this study, we used structure-function analysis to define the functional parameters of Ago2-mediated miRNA biogenesis. These include (1) the requirement for base-pairing at most, but not all, positions within the pre-mir-451 stem; (2) surprisingly little influence of the 5'-nucleotide on Ago sorting; (3) substantial influence of Ago protein stoichiometry on mir-451 maturation; (4) strong influence of G:C content in the distal stem on 3' resection of cleaved mir-451 substrates; and (5) the influence of hairpin length on substrate utilization by Ago2 and Dicer. Unexpectedly, we find that certain hairpin lengths confer competence to mature via both Dicer-mediated and Ago2-mediated pathways, and we show, in fact, that a conventional shRNA can traverse the Dicer-independent pathway. Altogether, these data inform the design of effective Dicer-independent substrates for gene silencing and reveal novel aspects of substrate handling by Ago proteins.
authors.affiliation: Department of Developmental Biology, Sloan-Kettering Institute, New York, New York 10065, USA.
authors.forename: Jr-Shiuan
authors.initials: JS
authors.lastname: Yang
authors.forename: Thomas
authors.initials: T
authors.lastname: Maurin
authors.forename: Eric C
authors.initials: EC
authors.lastname: Lai
mesh_terms: D060565:Argonaute Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001482:Base Composition; D020029:Base Pairing; D001483:Base Sequence; D002460:Cell Line; D006801:Humans; D055029:Inverted Repeat Sequences; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D060565:Argonaute Proteins; D035683:MicroRNAs; D043244:Ribonuclease III