Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production

Suresh K. Kandasamy, Ryuya Fukunaga

Research output: Contribution to journalArticlepeer-review

Abstract

The enzyme Dicer produces small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22-24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5'-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5'-monophosphate of a long dsRNA substrate is anchored by the phosphatebinding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing.

Original languageEnglish (US)
Pages (from-to)14031-14036
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume113
Issue number49
DOIs
StatePublished - Dec 6 2016

Keywords

  • Dicer
  • DsRNA
  • Phosphate
  • RNA silencing
  • SiRNA

ASJC Scopus subject areas

  • General

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