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: HSQC-TROSY experiments for the single domain (Supplementary Figure S2) and for the pair (Supplementary Figure S3) respectively were compared to assess the influence that the domains may have on the chemical shifts of each other. The superimposition of both spectra (Figure 2C) acquired under identical conditions reveals that the comparison of the amide resonances only matches reasonably well in certain areas. Significant differences in the chemical shift of many residues are observed between the RRM1 and the RRM1–RRM2 construct. The linker connecting both domains is very short in length (six residues), limiting the freedom of the domains in the tandem. The comparison of the linewidths (Supplementary Figure S4) of the independent RRM1 domain to that of the construct containing both domains indicates a clear broadening of the amide signals characteristic of a 23 KDa sample and that the RRM tandem behaves as a unit. Moreover, this observation is supported by the homogeneous T1, T2 and heteronuclear NOE values (Figure 3A, B and D respectively) measured for the tandem along its entire sequence. The calculated rotational correlation time (τc = 16 ns) is in agreement with the average value obtained for a globular protein of this size (Figure 3C) (28). This further confirms that the CPEB4 RRM1–RRM2 domains do not tumble independently. Based on these evidence, we interpret that the chemical shift changes observed in the RRM1 domain indicate the presence of a conformation where both RRM domains are close to one another. Furthermore, when mapping the most affected residues onto the homology model of the RRM1 domain, they cluster on one surface (Figure 2D), indicating that this surface should be in proximity of the RRM2 domain.
Affiliation: Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, Barcelona 08028, Spain.