Structural basis of siRNA recognition by TRBP double‐stranded RNA binding domains

• Published in: The EMBO journal Vol. 37; no. 6
• Main Authors: Masliah, Gregoire, Maris, Christophe, König, Sebastian LB, Yulikov, Maxim, Aeschimann, Florian, Malinowska, Anna L, Mabille, Julie, Weiler, Jan, Holla, Andrea, Hunziker, Juerg, Meisner‐Kober, Nicole, Schuler, Benjamin, Jeschke, Gunnar, Allain, Frederic H‐T
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 15-03-2018; Blackwell Publishing Ltd; John Wiley and Sons Inc
• Subjects: Asymmetry; Binding sites; Cleavage; Dicer; Double-stranded RNA; EMBO36; EMBO40; miRNA; NMR; Nuclear magnetic resonance; Nucleotide sequence; Recognition; Ribonuclease III; Ribonucleic acid; RNA; RNA-mediated interference; single‐molecule FRET; siRNA; Spectroscopy; TRBP; single‐molecule FRET; NMR; siRNA; TRBP; Dicer
• ISSN: 0261-4189; 1460-2075
• DOI: 10.15252/embj.201797089

The accurate cleavage of pre‐micro(mi)RNAs by Dicer and mi/siRNA guide strand selection are important steps in forming the RNA‐induced silencing complex (RISC). The role of Dicer binding partner TRBP in these processes remains poorly understood. Here, we solved the solution structure of the two N‐terminal dsRNA binding domains (dsRBDs) of TRBP in complex with a functionally asymmetric siRNA using NMR, EPR, and single‐molecule spectroscopy. We find that siRNA recognition by the dsRBDs is not sequence‐specific but rather depends on the RNA shape. The two dsRBDs can swap their binding sites, giving rise to two equally populated, pseudo‐symmetrical complexes, showing that TRBP is not a primary sensor of siRNA asymmetry. Using our structure to model a Dicer‐TRBP‐siRNA ternary complex, we show that TRBP's dsRBDs and Dicer's RNase III domains bind a canonical 19 base pair siRNA on opposite sides, supporting a mechanism whereby TRBP influences Dicer‐mediated cleavage accuracy by binding the dsRNA region of the pre‐miRNA during Dicer cleavage.
Synopsis
RNA‐binding protein TRBP associates with RNase Dicer to facilitate miRNA biogenesis. An NMR structure of TRBP's dsRNA binding domains shows that TRBP and Dicer bind pre‐miRNAs on opposite sides during processing, suggesting that TRBP works by increasing Dicer accuracy rather than strand selection.
The double‐stranded RNA binding domains (dsRBDs) of TRBP do not sense siRNA asymmetry.
The TRBP dsRBDs bind an asymmetric siRNA in two symmetric, equally populated, orientations.
There is no competition for pre‐miRNA binding between the TRBP dsRBDs and Dicer.
Graphical Abstract
NMR studies of the dsRNA‐binding protein TRBP suggest that its role in facilitating miRNA biogenesis is to increase accuracy of the Dicer RNase.