Distinguishable in vitro binding mode of monomeric TRBP and dimeric PACT with siRNA

PLoS One. 2013 May 2;8(5):e63434. doi: 10.1371/journal.pone.0063434. Print 2013.

Abstract

RNA interference (RNAi) is an evolutionally conserved posttranscriptional gene-silencing mechanism whereby small interfering RNA (siRNA) triggers sequence-specific cleavage of its cognate mRNA. Dicer, Argonaute (Ago), and either TAR-RNA binding protein (TRBP) or a protein activator of PKR (PACT) are the primary components of the RNAi pathway, and they comprise the core of a complex termed the RNA-induced silencing complex (RISC)-loading complex (RLC). TRBP and PACT share similar structural features including three dsRNA binding domains (dsRBDs), and a complex containing Dicer and either TRBP or PACT is considered to sense thermodynamic asymmetry of siRNA ends for guide strand selection. Thus, both TRBP and PACT are thought to participate in the RNAi pathway in an indistinguishable manner, but the differences in siRNA binding mode and the functional involvement of TRBP and PACT are poorly understood. Here, we show in vitro binding patterns of human TRBP and PACT to siRNA using electrophoresis mobility shift analysis and gel filtration chromatography. Our results clearly showed that TRBP and PACT have distinct in vitro siRNA binding patterns from each other. The results suggest that monomeric TRBP binds to siRNA at the higher affinity compared to the affinity for own homodimerization. In contrast, the affinity between PACT and siRNA is lower than that of homodimerization or that between TRBP and siRNA. Thus, siRNA may be more readily incorporated into RLC, interacting with TRBP (instead of PACT) in vivo.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Humans
  • Protein Binding
  • Protein Multimerization*
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • RNA Interference
  • RNA, Double-Stranded / genetics
  • RNA, Double-Stranded / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism*
  • RNA-Binding Proteins / chemistry*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Sequence Deletion

Substances

  • PRKRA protein, human
  • RNA, Double-Stranded
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • trans-activation responsive RNA-binding protein

Grant support

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Core Research Project for Private University: matching fund subsidy to K.U.-T. and a Research Fellowship for Young Scientists of the Japan Society for the Promotion of Science to T.T. The funders had no role in study, design, data collection and analysis, decision to publish, or preparation of the manuscript.