Functional parameters of Dicer-independent microRNA biogenesis

RNA. 2012 May;18(5):945-57. doi: 10.1261/rna.032938.112. Epub 2012 Mar 29.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Argonaute Proteins / chemistry
  • Argonaute Proteins / genetics
  • Argonaute Proteins / metabolism
  • Base Composition
  • Base Pairing
  • Base Sequence
  • Cell Line
  • Humans
  • Inverted Repeat Sequences
  • MicroRNAs / chemistry
  • MicroRNAs / genetics
  • MicroRNAs / metabolism*
  • Molecular Sequence Data
  • Mutation
  • Ribonuclease III / genetics
  • Ribonuclease III / metabolism*
  • Sequence Alignment

Substances

  • Argonaute Proteins
  • MicroRNAs
  • Ribonuclease III