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

Edwards, John; Long, Jed; Moor, Cornelia H. de; Emsley, Jonas; Searle, Mark S.

doi:10.1093/nar/gkt470

Cited by 29 publications

(22 citation statements)

References 79 publications

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“…Similar results were obtained when analyzing other top motifs for these two factors. Together, these data suggest that RBFOX2 and CELF1 preferentially recognize single-stranded RNA motifs and that intramolecular base-pairing directly competes with RBP recognition of these RNA motifs to a roughly similar extent for both proteins (Auweter et al, 2006; Edwards et al, 2013). …”

Section: Resultsmentioning

confidence: 78%

RNA Bind-n-Seq: Quantitative Assessment of the Sequence and Structural Binding Specificity of RNA Binding Proteins

et al. 2014

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Summary Specific protein-RNA interactions guide post-transcriptional gene regulation. Here we describe RNA Bind-n-Seq (RBNS), a method that comprehensively characterizes sequence and structural specificity of RNA binding proteins (RBPs), and its application to the developmental alternative splicing factors RBFOX2, CELF1/CUGBP1 and MBNL1. For each factor, we recovered both canonical motifs and additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in motifs containing UGC and/or GCU. Dissociation constants calculated from RBNS data using a novel algorithm correlated highly with values measured by surface plasmon resonance. Motifs identified by RBNS were conserved, were bound and active in vivo, and distinguished the subset of motifs enriched by CLIP-Seq that had regulatory activity. Together, our data demonstrate that RBNS complements crosslinking-based methods and show that in vivo binding and activity of these splicing factors is driven largely by intrinsic RNA affinity.

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Section: Resultsmentioning

confidence: 78%

RNA Bind-n-Seq: Quantitative Assessment of the Sequence and Structural Binding Specificity of RNA Binding Proteins

et al. 2014

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“…EDEN15 is a UG-rich RNA sequence known to bind CELF1 17 . Conventional biotinylated RNA pulldown 18 with EDEN15 yielded ~4-fold enrichment of CELF1 proteins over scrambled controls in HEK293T cells (Supplementary Fig.…”

Section: Validation Of Rapid With Known Rna–protein Interactionsmentioning

confidence: 99%

RNA–protein interaction detection in living cells

et al. 2018

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RNA–protein interactions play numerous roles in cellular function and disease. Here we describe RNA–protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA–protein interactions in living cells on a timescale as short as 1 min.

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“…To further establish that CELF1's ability to drive EMT is dependent on its RNA-binding activity, we designed RNA-binding mutants of CELF1 based on previously published structural work implicating five distinct residues distributed through CELF1's three RNA-recognition motifs (RRMs) as mediators of CELF1's RNA-binding functionality20. We generated a battery of individual RRM mutants (ΔD1, ΔD2, ΔD3) in which candidate amino-acid residues within each individual RRM were mutated to alanine, and a fourth mutant in which all five residues in the three RRMs were mutated in aggregate (ΔD1–3).…”

Section: Resultsmentioning

confidence: 99%

CELF1 is a central node in post-transcriptional regulatory programmes underlying EMT

et al. 2016

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The importance of translational regulation in tumour biology is increasingly appreciated. Here, we leverage polyribosomal profiling to prospectively define translational regulatory programs underlying epithelial-to-mesenchymal transition (EMT) in breast epithelial cells. We identify a group of ten translationally regulated drivers of EMT sharing a common GU-rich cis-element within the 3′-untranslated region (3′-UTR) of their mRNA. These cis-elements, necessary for the regulatory activity imparted by these 3′-UTRs, are directly bound by the CELF1 protein, which itself is regulated post-translationally during the EMT program. CELF1 is necessary and sufficient for both mesenchymal transition and metastatic colonization, and CELF1 protein, but not mRNA, is significantly overexpressed in human breast cancer tissues. Our data present an 11-component genetic pathway, invisible to transcriptional profiling approaches, in which the CELF1 protein functions as a central node controlling translational activation of genes driving EMT and ultimately tumour progression.

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

Cited by 29 publications

References 79 publications

RNA Bind-n-Seq: Quantitative Assessment of the Sequence and Structural Binding Specificity of RNA Binding Proteins

RNA Bind-n-Seq: Quantitative Assessment of the Sequence and Structural Binding Specificity of RNA Binding Proteins

RNA–protein interaction detection in living cells

CELF1 is a central node in post-transcriptional regulatory programmes underlying EMT

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