Molecular basis for asymmetry sensing of siRNAs by the Drosophila Loqs-PD/Dcr-2 complex in RNA interference

Abstract

RNA interference defends against RNA viruses and retro-elements within an organism's genome. It is triggered by duplex siRNAs, of which one strand is selected to confer sequence-specificity to the RNA induced silencing complex (RISC). In Drosophila, Dicer-2 (Dcr-2) and the double-stranded RNA binding domain (dsRBD) protein R2D2 form the RISC loading complex (RLC) and select one strand of exogenous siRNAs according to the relative thermodynamic stability of base-pairing at either end. Through genome editing we demonstrate that Loqs-PD, the Drosophila homolog of human TAR RNA binding protein (TRBP) and a paralog of R2D2, forms an alternative RLC with Dcr-2 that is required for strand choice of endogenous siRNAs in S2 cells. Two canonical dsRBDs in Loqs-PD bind to siRNAs with enhanced affinity compared to miRNA/miRNA* duplexes. Structural analysis, NMR and biophysical experiments indicate that the Loqs-PD dsRBDs can slide along the RNA duplex to the ends of the siRNA. A moderate but notable binding preference for the thermodynamically more stable siRNA end by Loqs-PD alone is greatly amplified in complex with Dcr-2 to initiate strand discrimination by asymmetry sensing in the RLC.

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

“…1c). Strong line broadening of all imino signals suggests dynamic binding involving sliding of the dsRBDs on the RNA helix, as previously shown for other dsRBDs 45,46,47 . With the shorter SRS2 stem-loop RNA, all imino signals are observable at equimolar RNA:protein ratio.…”

“…Sliding as initial binding mode also occurs in other RNA binding proteins such as the Drosophila protein Loqs-PD, a member of the siRNA silencing pathway. Loqs-PD contains two canonical dsRBDs that show highly dynamic binding and involves sliding along RNA stems 46 . A similar activity was also reported for the human ortholog of Loqs-PD, TAR RNA binding protein (TRBP) 45 .…”

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

“…The stability of the mi/siRNA duplex termini has been proposed to be one of the determinants of strand selection and is based on the observation that the RNA strand with the less stable 5 0 end is usually selected as the guide (Khvorova et al, 2003;Schwarz et al, 2003). The heterodimers R2D2/Dcr-2 and Loqs/Dcr-2 in flies, and Dicer/TRBP in humans, are able to bind asymmetric siRNA duplexes with a well-defined orientation (Tomari et al, 2004;Noland et al, 2011;Tants et al, 2017). It has also been shown that human Dicer is dispensable for asymmetric RISC assembly (Betancur & Tomari, 2012).…”

Conformational dynamics at microsecond timescale in the RNA-binding regions of dsRNA-binding domains

“…Three regions of the dsRBD namely, 1) middle of helix α 1 , 2) loop between β 1 and β 2 , and 3) N-terminal residues of the helix α 2 are involved in the interactions with the backbone of the dsRNA at its minor-major-minor grove spanning a length of ~12 bp. Most dsRBDs are known to interact with dsRNAs having A-form helical structure independent of the sequence (Masliah, Barraud and Allain, 2013;Tants et al, 2017). However, the target dsRNAs often have structural defects like internal mismatches/loops and bulges perturbing the A-form helical shape of the RNA.…”