RNA recognition and self-association of CPEB4 is mediated by its tandem RRM domains.
Bottom Line: 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.We propose a model of the CPEB4 RRM1-RRM2-CPE complex that illustrates the experimental data.
Affiliation: Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, Barcelona 08028, Spain.Show MeSH
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Mentions: Using this technique we could identify in the apo RRM1 sample (Figure 6A) and in the apo RRM1–RRM2 (Figure 6B) the presence of monomeric as well as dimeric species, consistent with the ITC results and the semi-native SDS PAGE. For the complex of RRM1 with RNA, a weak binding affinity was expected. Accordingly, we detected only a minor part of the species in a monomeric complex, the majority remained in its free form (Figure 6C). For the complex with the RRM-tandem domains, the presence of monomeric complexes as well as protein dimers bound to two ligands was detected (Figure 6D). Moreover, no unbound species were detected. The different species (see schematic representation in Figure 6E) were unambiguously identified based on their specific mass-to-charge ratio (MS) and/or their characteristic drift-times that correlate their size-to-charge ratio (IM). Therefore, IM-MS confirms the hypothesis that indeed dimeric species are present. Perhaps, the presence of monomeric and dimeric forms renders the determination of the protein concentration in native conditions inaccurate, explaining the stoichiometry values inferior to one. The results also clearly indicate that only 1:1 protein–RNA complexes are present in both the monomeric and dimeric forms (ML, D2L). The presence of the M1L and D2L species probably contributes to the global affinity measured by the ITC experiments, which is mainly governed by the contribution of the M1L form. The results of the IM-MS experiments with RRM1–RRM2 in complex with RNA reveal that the presence of the ligand does not prevent the formation of dimeric species and does not render the RNA binding site inaccessible. We therefore conclude that the dimerization surface of the RRM1–RRM2 is separate from its RNA-binding interface.
Affiliation: Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, Barcelona 08028, Spain.