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Multiple sensors ensure guide strand selection in human RNAi pathways.

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  • 1Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.

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|March 28, 2013
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Human Argonaute2 (Ago2) protein selects RNA strands for RNA interference (RNAi), with help from Dicer and dsRBPs like TRBP or PACT. Strand selection depends on RNA features and the specific protein complex involved.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Small RNAs regulate gene expression via RNA interference (RNAi) by guiding RNA-induced silencing complexes (RISCs).
  • Accurate target recognition by RISC relies on the preferential loading of one RNA strand (guide strand).
  • The mechanisms governing guide strand selection in human RNAi are not fully understood.

Purpose of the Study:

  • To investigate the roles of core human RNAi machinery components in RNA guide strand selection.
  • To determine how Argonaute2 (Ago2), Dicer, and double-stranded RNA-binding proteins (dsRBPs) like TRBP and PACT contribute to this process.
  • To identify factors influencing strand selection specificity for siRNAs and miRNAs.

Main Methods:

  • Utilized a reconstituted in vitro system to study human RNAi components.
  • Assessed the intrinsic RNA strand selection capability of Argonaute2 (Ago2).
  • Examined the impact of Dicer and dsRBP (TRBP, PACT) complex formation on Ago2's strand selection activity.

Main Results:

  • Ago2 exhibits intrinsic, albeit substrate-dependent, RNA strand selection ability.
  • Dicer/dsRBP complexes, particularly those with TRBP or PACT, significantly enhance Ago2's strand selection.
  • Strand selection efficiency is influenced by siRNA/miRNA duplex thermodynamics, 5' nucleotide identity, and structure.
  • PACT-containing complexes enhance miRNA strand selection differently than TRBP-containing complexes.
  • TRBP, but not PACT, appears to disfavor overall mRNA targeting by miRNAs within complexes.

Conclusions:

  • Multiple proteins collaborate to ensure efficient and accurate guide strand selection in the human RNAi pathway.
  • The specific dsRBPs (TRBP or PACT) associated with Dicer/Ago2 complexes can influence both strand selection and downstream mRNA targeting.
  • These findings suggest distinct RNAi pathway mechanisms may exist based on the presence of TRBP or PACT.