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Sequence determinants for the tandem recognition of UGU and CUG rich RNA elements by the two N—terminal RRMs of CELF1

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 t...

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Autores principales: Edwards, John, Malaurie, Emilie, Kondrashov, Alexander, Long, Jed, de Moor, Cornelia H., Searle, Mark S., Emsley, Jonas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3201864/
https://www.ncbi.nlm.nih.gov/pubmed/21743084
http://dx.doi.org/10.1093/nar/gkr510
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author Edwards, John
Malaurie, Emilie
Kondrashov, Alexander
Long, Jed
de Moor, Cornelia H.
Searle, Mark S.
Emsley, Jonas
author_facet Edwards, John
Malaurie, Emilie
Kondrashov, Alexander
Long, Jed
de Moor, Cornelia H.
Searle, Mark S.
Emsley, Jonas
author_sort Edwards, John
collection PubMed
description 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.
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spelling pubmed-32018642011-10-26 Sequence determinants for the tandem recognition of UGU and CUG rich RNA elements by the two N—terminal RRMs of CELF1 Edwards, John Malaurie, Emilie Kondrashov, Alexander Long, Jed de Moor, Cornelia H. Searle, Mark S. Emsley, Jonas Nucleic Acids Res Structural Biology 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. Oxford University Press 2011-10 2011-07-09 /pmc/articles/PMC3201864/ /pubmed/21743084 http://dx.doi.org/10.1093/nar/gkr510 Text en © The Author(s) 2011. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Structural Biology
Edwards, John
Malaurie, Emilie
Kondrashov, Alexander
Long, Jed
de Moor, Cornelia H.
Searle, Mark S.
Emsley, Jonas
Sequence determinants for the tandem recognition of UGU and CUG rich RNA elements by the two N—terminal RRMs of CELF1
title Sequence determinants for the tandem recognition of UGU and CUG rich RNA elements by the two N—terminal RRMs of CELF1
title_full Sequence determinants for the tandem recognition of UGU and CUG rich RNA elements by the two N—terminal RRMs of CELF1
title_fullStr Sequence determinants for the tandem recognition of UGU and CUG rich RNA elements by the two N—terminal RRMs of CELF1
title_full_unstemmed Sequence determinants for the tandem recognition of UGU and CUG rich RNA elements by the two N—terminal RRMs of CELF1
title_short Sequence determinants for the tandem recognition of UGU and CUG rich RNA elements by the two N—terminal RRMs of CELF1
title_sort sequence determinants for the tandem recognition of ugu and cug rich rna elements by the two n—terminal rrms of celf1
topic Structural Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3201864/
https://www.ncbi.nlm.nih.gov/pubmed/21743084
http://dx.doi.org/10.1093/nar/gkr510
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