Characterization of endogenous human Argonautes and their miRNA partners in RNA silencing

Asuka Azuma-Mukai, Hideo Oguri, Toutai Mituyama, Rong Qian Zhi, Kiyoshi Asai, Haruhiko Siomi, Mikiko C. Siomi

Research output: Contribution to journalArticle

188 Citations (Scopus)

Abstract

Small RNAs triggering RNA silencing are loaded onto Argonautes and then sequence-specifically guide them to target transcripts. Epitope-tagged human Argonautes (hAgo1, hAgo2, hAgo3, and hAgo4) are associated with siRNAs and miRNAs, but only epitope-tagged hAgo2 has been shown to have Slicer activity. Contrarily, how endogenous hAgos behave with respect to small RNA association and target RNA destruction has remained unclear. Here, we produced monoclonal antibodies for individual hAgos. High-throughput pyrosequencing revealed that immunopurified endogenous hAgo2 and hAgo3 associated mostly with miRNAs. Endogenous hAgo3 did not show Slicer function but localized in P-bodies, suggesting that hAgo3 endogenously expressed is, like hAgo2, involved in the miRNA pathway but antagonizes the RNAi activity of hAgo2. Sequence variations of miRNAs were found at both 5′ and 3′ ends, suggesting that multiple mature miRNAs containing different "seed" sequences can arise from one miRNA precursor. The hAgo antibodies we raised are valuable tools for ascertaining the functional behavior of endogenous Argonautes and miRNAs in RNA silencing.

Original languageEnglish
Pages (from-to)7964-7969
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number23
DOIs
Publication statusPublished - 2008 Jun 10

Keywords

  • Jurkat
  • Slicer
  • siRNAs

ASJC Scopus subject areas

  • General

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