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

Abstract

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.

The crystal structure of Staufen1 in complex with a physiological RNA sheds light on substrate selectivity

“…Both dsRBD3 molecules in our structure engage all three regions in RNA binding ( Fig 4A ). This binding mode is similar to what was observed for dStau dsRBD3 ( Ramos et al, 2000 ) and other dsRBD–dsRNA complexes ( Ryter & Schultz, 1998 ; Blaszczyk et al, 2004 ; Wu et al, 2004 ; Gan et al, 2006 , 2008 ; Stefl et al, 2006 , 2010 ; Wang et al, 2011 ; Jayachandran et al, 2016 ; Masliah et al, 2018 ). Conversely, dsRBD4 primarily uses regions 1 and 3 for binding, whereas the β1–β2 loop in region 2 is partially unstructured and does not engage in close interactions with the RNA ( Fig 4A ).…”

Staufen2-mediated RNA recognition and localization requires combinatorial action of multiple domains

“…Thus, a stem of 8 bp appears to be the minimal length required for recognition by mStau2 tandem domains. Of note, available structures of dsRBDs show binding to longer RNA stem loop of about 19 bp length 30,31 . It is therefore well possible that our observed binding to a minimal stem-loop RNA only reflects a partial recognition and that for a full binding a longer stem is required.…”

Molecular basis for AU-rich element recognition and dimerization by the HuR C-terminal RRM

“…The c-fos ARE 11-mer (5′-AUUUUUAUUUU-3′) and the 11-nt A-rich RNA were provided by J. Hunziger, Novartis, and synthesized as described (53). Short single-stranded RNAs (5′-UUUUU-, -AUUUU-, -UUAUU-, -AUUAU-, -UUUUA-, -UUUUUU-, -AUUUUU-, -UUUAUU-, -AUUAUU-3′) were purchased from Thermo Fisher Scientific and deprotected as described in the manufacturer’s instructions, lyophilized, and dissolved in NMR or ITC buffer.…”