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.

Functional Integration of mRNA Translational Control Programs

“…No association of Musashi1 was observed with GST tagged BRaf (an activator of MAP kinase signaling, [ 87 ]), demonstrating specificity of the Musashi1:Musashi1 and Musashi1:CPEB interactions. Our analyses also revealed CPEB1:CPEB1 and CPEB4:CPEB4 homodimer formation ( Figure 3 B, left and right panels, respectively), consistent with prior findings [ 88 , 89 ]. Interestingly, we also observed CPEB1:CPEB4 heterodimers ( Figure 3 C), further adding to the range and complexity of mRNP formation between distinct RBPs.…”

Structural Analysis of the Pin1-CPEB1 interaction and its potential role in CPEB1 degradation

“…In order to characterize the presence of complexes of different stoichiometries, we applied Ion Mobility-Mass Spectrometry (IM-MS) since this technique is valuable to detect the populations of different protein complexes at concentrations used in ITC experiments 7 16 . The analysis of the IM-MS data revealed the presence of monomeric species in the apo Pin1 form ( Fig.…”

“…CPEBs also contain a protein-protein interaction site located at the N-terminus, which is highly divergent among the different members of the family. The RNA binding characteristics of CPEB1 and CPEB4 have been recently characterized to atomic detail 7 8 . The structural information has revealed that efficient recognition of the CPE sites is achieved using the pair of RRM domains working as a team, increasing the specificity by augmenting the protein-binding surface.…”

Regulation of the Expression of Cytoplasmic Polyadenylation Element Binding Proteins for the Treatment of Cancer

“…Of these RNA-binding proteins, cytoplasmic polyadenylation element binding protein (CPEB) family are sequence-specific RNA-binding proteins and the key factors controling the elongation of the poly(A) tail and polyadenylation-induced translation (2,5,6). CPEB binds the cytoplasmic polyadenylation element (CPE) in the 3' untranslated regions of responding mRNAs (2,(7)(8)(9). CPEB-mediated effects on translation require at least two CPEs separated by less than 50 nucleotides in the target mRNA, which indicates the formation of CPEB-dimer (10).…”

“…All CPEB proteins have a similar structure with the C-terminal regions composed of two RNA recognition motifs (RRMs) and two zinc-fingercontaining sequence-specific RNA-binding proteins, as well as a regulatory N-terminal region (1,9,12). The N-terminals of CPEB1-4 are highly variable, whereas the C-terminals are more conserved (1).…”