title: RNA recognition and self-association of CPEB4 is mediated by its tandem RRM domains.
abstract: Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation element binding proteins (CPEB) with cytoplasmic polyadenylation element (CPE) containing mRNAs. The CPEB family comprises four paralogs, CPEB1-4, each composed of a variable N-terminal region, two RNA recognition motif (RRM) and a C-terminal ZZ-domain. We have characterized the RRM domains of CPEB4 and their binding properties using a combination of biochemical, biophysical and NMR techniques. Isothermal titration calorimetry, NMR and electrophoretic mobility shift assay experiments demonstrate that both the RRM domains are required for an optimal CPE interaction and the presence of either one or two adenosines in the two most commonly used consensus CPE motifs has little effect on the affinity of the interaction. Both the single RRM1 and the tandem RRM1-RRM2 have the ability to dimerize, although representing a minor population. Self-association does not affect the proteins' ability to interact with RNA as demonstrated by ion mobility-mass spectrometry. Chemical shift effects measured by NMR of the apo forms of the RRM1-RRM2 samples indicate that the two domains are orientated toward each other. NMR titration experiments show that residues on the β-sheet surface on RRM1 and at the C-terminus of RRM2 are affected upon RNA binding. We propose a model of the CPEB4 RRM1-RRM2-CPE complex that illustrates the experimental data.
authors: Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, Barcelona 08028, Spain.
authors: Constanze
authors: C
authors: Schelhorn
authors: Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, Barcelona 08028, Spain.
authors: James M B
authors: JM
authors: Gordon
authors: Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, Barcelona 08028, Spain.
authors: Lidia
authors: L
authors: Ruiz
authors: Departament de Química Orgànica and IBUB, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain.
authors: Javier
authors: J
authors: Alguacil
authors: Departament de Química Orgànica and IBUB, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain.
authors: Enrique
authors: E
authors: Pedroso
authors: Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, Barcelona 08028, Spain Catalan Institution for Research and Advanced Studies (ICREA), Passeig Lluis Companys 23, Barcelona 08010, Spain maria.macias@irbbarcelona.org.
authors: Maria J
authors: MJ
authors: Macias
mesh_terms: D001665:Binding Sites; D006801:Humans; D008958:Models, Molecular; D059372:Nucleotide Motifs; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C554939:CPEB4 protein, human; D016601:RNA-Binding Proteins; D012313:RNA