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11:34
Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins
Published on: August 9, 2019
CUG-BP1/CELF1 requires UGU-rich sequences for high-affinity binding
Julien Marquis1, Luc Paillard, Yann Audic
1Généthon, CNRS UMR 8115, 1 bis rue de l'Internationale, 91002 Evry cedex 2, France.
The Biochemical Journal
|August 30, 2006
Summary
CUG-binding protein 1 (CUG-BP1) and its Xenopus homologue EDEN-BP bind specific RNA sequences. High-affinity binding sites, rich in UGU motifs, target mRNA for deadenylation, revealing CUG-BP1
Area of Science:
- Molecular Biology
- RNA Biology
- Developmental Biology
Background:
- CUG-binding protein 1 (CUG-BP1) is a human RNA-binding protein involved in RNA processing.
- Its Xenopus homologue, EDEN-BP, is crucial for maternal mRNA deadenylation post-fertilization.
- The precise binding specificity of these proteins remains under investigation.
Purpose of the Study:
- To characterize the RNA binding specificity of CUG-BP1.
- To identify sequence elements that mediate high-affinity binding and functional deadenylation.
- To investigate the role of CUG-BP1/EDEN-BP in regulating maternal mRNA stability.
Main Methods:
- Systematic evolution of ligand by exponential enrichment (SELEX) to select RNA aptamers binding CUG-BP1.
- Surface plasmon resonance and electrophoretic mobility-shift assays to assess binding affinity and complex formation.
- In vivo deadenylation assays using reporter mRNA to evaluate functional consequences of aptamer binding.
- Bioinformatic analysis to identify sequence characteristics of high-affinity binding sites.
Main Results:
- Two families of aptamers with differential binding affinities for CUG-BP1 were selected.
- High-affinity aptamers formed distinct complexes with CUG-BP1 compared to low-affinity aptamers.
- Only high-affinity aptamers conferred deadenylation on reporter mRNA in vivo.
- High-affinity binding sites are characterized by UGU trinucleotide motifs and are at least 30 nucleotides long.
- The MAPK phosphatase (XCl100alpha) mRNA was identified as an EDEN-BP substrate.
Conclusions:
- High-affinity CUG-BP1 binding sites are defined by specific sequence features, including UGU motifs.
- These sites are functionally competent to direct mRNA deadenylation in vivo.
- The findings elucidate the molecular mechanisms underlying CUG-BP1/EDEN-BP-mediated RNA regulation during early development.
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