Conserved asymmetry underpins homodimerization of Dicer-associated double-stranded RNA-binding proteins.

Alex Heyam,Claire E Coupland,Michael P Williamson,Michael J Plevin,Ruth A Haley,Dimitris Lagos,Pedro M Aguiar,Nicola J Baxter,Leonhard Jakob,Gunter Meister,Clément Dégut

doi:10.1093/nar/gkx928

Abstract

Double-stranded RNA-binding domains (dsRBDs) are commonly found in modular proteins that interact with RNA. Two varieties of dsRBD exist: canonical Type A dsRBDs interact with dsRNA, while non-canonical Type B dsRBDs lack RNA-binding residues and instead interact with other proteins. In higher eukaryotes, the microRNA biogenesis enzyme Dicer forms a 1:1 association with a dsRNA-binding protein (dsRBP). Human Dicer associates with HIV TAR RNA-binding protein (TRBP) or protein activator of PKR (PACT), while Drosophila Dicer-1 associates with Loquacious (Loqs). In each case, the interaction involves a region of the protein that contains a Type B dsRBD. All three dsRBPs are reported to homodimerize, with the Dicer-binding region implicated in self-association. We report that these dsRBD homodimers display structural asymmetry and that this unusual self-association mechanism is conserved from flies to humans. We show that the core dsRBD is sufficient for homodimerization and that mutation of a conserved leucine residue abolishes self-association. We attribute differences in the self-association properties of Loqs, TRBP and PACT to divergence of the composition of the homodimerization interface. Modifications that make TRBP more like PACT enhance self-association. These data are examined in the context of miRNA biogenesis and the protein/protein interaction properties of Type B dsRBDs.

Highlights

Double-stranded RNA-binding domains are found in all domains of life, and contribute to diverse biological processes ranging from splicing to antiviral responses [1,2]
We show that asymmetry results from the formation of an inter-molecular parallel ␤-sheet, which is stabilized by an inter-strand hydrogen bonding network that would not be possible in a symmetric parallel dimer
Codon-optimized sequences of PACT and TAR RNA-binding protein (TRBP) were ordered from GeneArt, and regions corresponding to PACT residues 239–313 (PACT-dsRBD 3 (D3)) and 208–313 (PACT-Ext-D3), and TRBP residues 258–366 (TRBP-Ext-D3) were cloned into a vector derived from pET-28a [24] using an In-Fusion cloning strategy (Clontech)

Summary

Introduction

Double-stranded (ds) RNA-binding domains (dsRBDs; called dsRNA-binding motifs or dsRBMs) are found in all domains of life, and contribute to diverse biological processes ranging from splicing to antiviral responses [1,2]. All dsRBDs adopt a common ␣-␤-␤-␤-␣ fold, but they can be divided into two distinct classes: those that bind dsRNA and those that do not [3]. Type-A dsRBDs bind dsRNA via three conserved regions. This interaction rarely displays any specificity for RNA sequence and is instead dependent on dsRNA-specific groove structures and the 2′-OH of the ribose sugar [1]. Type B dsRBDs lack residue conservation in dsRNA recognition regions, including two critical lysine residues in dsRNA recognition Region 3, and cannot bind dsRNA [3]. Type B dsRBDs have evolved to mediate protein–protein interactions. While there have been many structural studies of Type A dsRBDs, only recently has structural information about protein–protein interactions mediated by Type B domains become available

Methods

Results

Conclusion