Structural insights into the targeting of mRNA GU-rich elements by the three RRMs of CELF1

J M Edwards,J Emsley,C H De Moor,J Long,M S Searle

doi:10.1093/nar/gkt470

Abstract

The CUG-BP, Elav-like family (CELF) of RNA-binding proteins control gene expression at a number of different levels by regulating pre-mRNA splicing, deadenylation and mRNA stability. We present structural insights into the binding selectivity of CELF member 1 (CELF1) for GU-rich mRNA target sequences of the general form 5′-UGUNxUGUNyUGU and identify a high affinity interaction (Kd ∼ 100 nM for x = 2 and y = 4) with simultaneous binding of all three RNA recognition motifs within a single 15-nt binding element. RNA substrates spin-labelled at either the 3′ or 5′ terminus result in differential nuclear magnetic resonance paramagnetic relaxation enhancement effects, which are consistent with a non-sequential 2-1-3 arrangement of the three RNA recognition motifs on UGU sites in a 5′ to 3′ orientation along the RNA target. We further demonstrate that CELF1 binds to dispersed single-stranded UGU sites at the base of an RNA hairpin providing a structural rationale for recognition of CUG expansion repeats and splice site junctions in the regulation of alternative splicing.

Highlights

The CELF family of RNA-binding proteins, which includes CELF member 1 (CELF1) (CUG-BP1) in humans, ETR-3-like factors, embryonic deadenylation element binding protein (EDEN-BP) in Xenopus and the highly homologous Bruno in Drosophila [1,2,3,4,5], are widely conserved in nature and regulate multiple facets of gene expression at the level of alternative splicing of mRNA, through translational regulation, deadenylation and mRNA stability [6,7,8,9,10,11,12,13]
Using a stable three-domain CELF1 construct (RRM123), we describe nuclear magnetic resonance (NMR) structural and biophysical studies based on chemical shift perturbation (CSP) analysis, paramagnetic relaxation enhancement (PRE) experiments with spinlabelled RNA substrates and binding affinity studies by isothermal titration calorimetry (ITC)
His-tagged bacterial expression constructs were produced for the individual CELF1 RNA recognition motifs (RRMs) 1, 2 and 3, for a fragment containing domains 1 and 2 (RRM12) and for the full-length 489 residue CELF1 protein using methodology previously described [33]

Summary

Introduction

The CELF family of RNA-binding proteins, which includes CELF1 (CUG-BP1) in humans, ETR-3-like factors, embryonic deadenylation element binding protein (EDEN-BP) in Xenopus and the highly homologous Bruno in Drosophila [1,2,3,4,5], are widely conserved in nature and regulate multiple facets of gene expression at the level of alternative splicing of mRNA, through translational regulation, deadenylation and mRNA stability [6,7,8,9,10,11,12,13]. Recent studies, using in vitro assays for assessing mRNA decay in AU-rich mRNAs, have shown that CELF1 binds to mRNAs and stimulates poly(A) shortening in the 30 untranslated region (30 UTR) by recruiting the poly(A) deadenylase ribonuclease (PARN) [13,30]. In this case, binding to target EDEN mRNA GU-rich elements (GREs) is necessary for function [2,12,31,32,33,34,35], with EDEN-dependent deadenylation regulated by neighbouring cis-acting AU-rich elements (AREs) [13]. These long-range interactions have been reported to be mediated through an interaction between CELF2 and the U2 snRNP, but this interaction appears to be absent for CELF1 [38]

Methods

Results

Conclusion