title: Sequence determinants for the tandem recognition of UGU and CUG rich RNA elements by the two N--terminal RRMs of CELF1.
abstract: CUGBP, Elav-like family member 1 (CELF1) is an RNA binding protein with important roles in the regulation of splicing, mRNA decay and translation. CELF1 contains three RNA recognition motifs (RRMs). We used gel retardation, gel filtration, isothermal titration calorimetry and NMR titration studies to investigate the recognition of RNA by the first two RRMs of CELF1. NMR shows that RRM1 is promiscuous in binding to both UGU and CUG repeat sequences with comparable chemical shift perturbations. In contrast, RRM2 shows greater selectivity for UGUU rather than CUG motifs. A construct (T187) containing both binding domains (RRM1 and RRM2) was systematically studied for interaction with tandem UGU RNA binding sites with different length linker sequences UGU(U)(x)UGU where x = 1-7. A single U spacer results in interactions only with RRM1, demonstrating both steric constraints in accommodating both RRMs simultaneously at adjacent sites, and also subtle differences in binding affinities between RRMs. However, high affinity co-operative binding (K(d) ~ 0.4 µM) is evident for RNA sequences with x = 2-4, but longer spacers (x ≥ 5) lead to a 10-fold reduction in affinity. Our analysis rationalizes the high affinity interaction of T187 with the 11mer GRE consensus regulatory sequence UGUUUGUUUGU and has significant consequences for the prediction of CELF1 binding sites.
authors: School of Chemistry, Centre for Biomolecular Sciences, University Park, Nottingham NG7 2RD, UK.
authors: John
authors: J
authors: Edwards
authors: Emilie
authors: E
authors: Malaurie
authors: Alexander
authors: A
authors: Kondrashov
authors: Jed
authors: J
authors: Long
authors: Cornelia H
authors: CH
authors: de Moor
authors: Mark S
authors: MS
authors: Searle
authors: Jonas
authors: J
authors: Emsley
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D024202:Electrophoretic Mobility Shift Assay; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D014466:Ultraviolet Rays; D029867:Xenopus Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C568810:CELF1 protein, Xenopus; D016601:RNA-Binding Proteins; D029867:Xenopus Proteins; D012313:RNA