Distinguishable In Vitro Binding Mode of Monomeric TRBP and Dimeric PACT with siRNA

Tomoko Takahashi,Kumiko Ui-Tei,Kenji Nishi,Shuhei Zenno,Takuya Miyakawa,Masaru Tanokura,Andreas Hofmann

doi:10.1371/journal.pone.0063434

Tomoko Takahashi, Kumiko Ui-Tei + Show 5 more

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https://doi.org/10.1371/journal.pone.0063434

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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.

Highlights

RNA interference (RNAi) and related pathways are evolutionally conserved mechanisms whereby single-stranded guide RNA triggers sequence-specific cleavage or translational repression of its cognate mRNA through the RNA-induced silencing complex (RISC) [1,2,3]
Our results clearly show that TARRNA binding protein (TRBP)-WT and PACT-WT proteins bind small interfering RNA (siRNA), their binding patterns based on electrophoresis mobility shift analysis (EMSA) differed (Figures 1B, 4B, S1A, C-F and S4A)
We revealed that PACT-dsRBDmt1+2 showed little binding activities to siRNA (Figures 5D and S4F), indicating that PACT proteins bind siRNA with dsRBD1 and dsRBD2, while dsRBD3 is not involved in siRNA binding

Summary

Introduction

RNA interference (RNAi) and related pathways are evolutionally conserved mechanisms whereby single-stranded guide RNA triggers sequence-specific cleavage or translational repression of its cognate mRNA through the RNA-induced silencing complex (RISC) [1,2,3]. Silencing is initiated by long double-stranded RNA (dsRNA) or hairpin-structured precursor-microRNA (premiRNA), which are processed by the RNaseIII enzyme Dicer to produce 21–23 nucleotide (nt) short interfering RNAs (siRNAs) or microRNAs (miRNAs), respectively [4,5,6] These small RNAs are loaded onto Argonaute (Ago2), the slicer of target mRNA [7], [8]. In human cells, both Dicer and the trans-activation response (TAR) RNA binding protein (TRBP) or the protein activator of protein kinase R (PKR) (PACT) are associated with RISC loading [8,9,10]. TRBP and PACT are known to interact with each other and associate with Dicer to facilitate dsRNA cleavage [14], [15]

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