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RNA recognition and self-association of CPEB4 is mediated by its tandem RRM domains.

Schelhorn C, Gordon JM, Ruiz L, Alguacil J, Pedroso E, Macias MJ - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, Barcelona 08028, Spain.

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(A) Superimposition of 1H, 15N-HSQC spectra of RRM1–RRM2 free (blue) and in complex with U5A2U1 (yellow) 1.5 molar equivalents. Zooms in various regions of the spectra are displaying all titration points. Spectra are colored as blue (free); orange, 0.5; dark red, 1.0, and yellow, 1.5 molar equivalents. (B) Chemical shift perturbations of RRM1–RRM2 domains upon binding to U5A2U1 (blue) and U5A1U2 (orange). The Chemical Shift Perturbations (CSPs) are plotted versus residue numbers. The dashed line in gray shows a cut-off Δδ > 0.1 ppm (average chemical shift perturbation over all resonances + standard deviation). The CSPs for both RNA ligands do not show any significant differences. Within RRM1, the largest shift perturbations are found in residues that are supposed to lie on the β-sheet; for RRM2, however, they clearly cluster in the C-terminal part. The RNP2 of RRM2 shows shifts just above the cut-off, which suggests that RNP2 is not the main binding site for RRM2, instead the C-terminal positively charged residues seem to play an essential role.
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Figure 5: (A) Superimposition of 1H, 15N-HSQC spectra of RRM1–RRM2 free (blue) and in complex with U5A2U1 (yellow) 1.5 molar equivalents. Zooms in various regions of the spectra are displaying all titration points. Spectra are colored as blue (free); orange, 0.5; dark red, 1.0, and yellow, 1.5 molar equivalents. (B) Chemical shift perturbations of RRM1–RRM2 domains upon binding to U5A2U1 (blue) and U5A1U2 (orange). The Chemical Shift Perturbations (CSPs) are plotted versus residue numbers. The dashed line in gray shows a cut-off Δδ > 0.1 ppm (average chemical shift perturbation over all resonances + standard deviation). The CSPs for both RNA ligands do not show any significant differences. Within RRM1, the largest shift perturbations are found in residues that are supposed to lie on the β-sheet; for RRM2, however, they clearly cluster in the C-terminal part. The RNP2 of RRM2 shows shifts just above the cut-off, which suggests that RNP2 is not the main binding site for RRM2, instead the C-terminal positively charged residues seem to play an essential role.

Mentions: The binding ligands used for ITC experiments were two octamer RNA fragments containing the two consensus CPE motifs, U5A2U1 and U5A1U2. ITC experiments with RRM1 and both ligands showed low-affinity binding with dissociation constants in the high μM range. The experiments with RRM1–RRM2 as titrate yielded dissociation constants of 323 ± 34 nM for U5A2U1 and 299 ± 28 nM for U5A1U2 (Figure 4B). Both ligands, which differ only in one nucleotide, show very similar KD values (both lying within the error limit of each other), thus not allowing us to significantly distinguish between the two binding affinities. The affinity increase due to the presence of the RRM2 domain is about 100-fold with respect to the values obtained for the single RRM1 domain. As we have not been able to obtain pure RRM2, the experiment could not be repeated for the single RRM2. However, from our NMR titration data, which allows the identification of the regions affected by RNA binding (Figure 5), it is clear that chemical shift perturbations are observed for residues in both domains. The titration of the RRM1 with U5A2U1 is shown in Supplementary Figure S5. Therefore, our data indicate that both RRM domains are important to maintain optimal RNA-binding activity and affinity. The improvement of the affinity by 100 times in the construct containing the RRM2 domain is quite remarkable since this domain contains a degenerate RNP motif and it was assumed to be a poor RNA binder. This increment in the affinity together with the length of the RNA recognized by the RRM1–RRM2 pair suggests that both RRM domains act cooperatively resulting in high-affinity binding of the RNA. We suggest that the role of the RRM2 in the RNA interaction is dual: it increases the global surface of the pair to recognize the RNA with respect to the single RRM1 and helps adjusting the relative orientation of the two domains to best accommodate the RNA ligand.


RNA recognition and self-association of CPEB4 is mediated by its tandem RRM domains.

Schelhorn C, Gordon JM, Ruiz L, Alguacil J, Pedroso E, Macias MJ - Nucleic Acids Res. (2014)

(A) Superimposition of 1H, 15N-HSQC spectra of RRM1–RRM2 free (blue) and in complex with U5A2U1 (yellow) 1.5 molar equivalents. Zooms in various regions of the spectra are displaying all titration points. Spectra are colored as blue (free); orange, 0.5; dark red, 1.0, and yellow, 1.5 molar equivalents. (B) Chemical shift perturbations of RRM1–RRM2 domains upon binding to U5A2U1 (blue) and U5A1U2 (orange). The Chemical Shift Perturbations (CSPs) are plotted versus residue numbers. The dashed line in gray shows a cut-off Δδ > 0.1 ppm (average chemical shift perturbation over all resonances + standard deviation). The CSPs for both RNA ligands do not show any significant differences. Within RRM1, the largest shift perturbations are found in residues that are supposed to lie on the β-sheet; for RRM2, however, they clearly cluster in the C-terminal part. The RNP2 of RRM2 shows shifts just above the cut-off, which suggests that RNP2 is not the main binding site for RRM2, instead the C-terminal positively charged residues seem to play an essential role.
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Related In: Results  -  Collection

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Figure 5: (A) Superimposition of 1H, 15N-HSQC spectra of RRM1–RRM2 free (blue) and in complex with U5A2U1 (yellow) 1.5 molar equivalents. Zooms in various regions of the spectra are displaying all titration points. Spectra are colored as blue (free); orange, 0.5; dark red, 1.0, and yellow, 1.5 molar equivalents. (B) Chemical shift perturbations of RRM1–RRM2 domains upon binding to U5A2U1 (blue) and U5A1U2 (orange). The Chemical Shift Perturbations (CSPs) are plotted versus residue numbers. The dashed line in gray shows a cut-off Δδ > 0.1 ppm (average chemical shift perturbation over all resonances + standard deviation). The CSPs for both RNA ligands do not show any significant differences. Within RRM1, the largest shift perturbations are found in residues that are supposed to lie on the β-sheet; for RRM2, however, they clearly cluster in the C-terminal part. The RNP2 of RRM2 shows shifts just above the cut-off, which suggests that RNP2 is not the main binding site for RRM2, instead the C-terminal positively charged residues seem to play an essential role.
Mentions: The binding ligands used for ITC experiments were two octamer RNA fragments containing the two consensus CPE motifs, U5A2U1 and U5A1U2. ITC experiments with RRM1 and both ligands showed low-affinity binding with dissociation constants in the high μM range. The experiments with RRM1–RRM2 as titrate yielded dissociation constants of 323 ± 34 nM for U5A2U1 and 299 ± 28 nM for U5A1U2 (Figure 4B). Both ligands, which differ only in one nucleotide, show very similar KD values (both lying within the error limit of each other), thus not allowing us to significantly distinguish between the two binding affinities. The affinity increase due to the presence of the RRM2 domain is about 100-fold with respect to the values obtained for the single RRM1 domain. As we have not been able to obtain pure RRM2, the experiment could not be repeated for the single RRM2. However, from our NMR titration data, which allows the identification of the regions affected by RNA binding (Figure 5), it is clear that chemical shift perturbations are observed for residues in both domains. The titration of the RRM1 with U5A2U1 is shown in Supplementary Figure S5. Therefore, our data indicate that both RRM domains are important to maintain optimal RNA-binding activity and affinity. The improvement of the affinity by 100 times in the construct containing the RRM2 domain is quite remarkable since this domain contains a degenerate RNP motif and it was assumed to be a poor RNA binder. This increment in the affinity together with the length of the RNA recognized by the RRM1–RRM2 pair suggests that both RRM domains act cooperatively resulting in high-affinity binding of the RNA. We suggest that the role of the RRM2 in the RNA interaction is dual: it increases the global surface of the pair to recognize the RNA with respect to the single RRM1 and helps adjusting the relative orientation of the two domains to best accommodate the RNA ligand.

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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, Barcelona 08028, Spain.

Show MeSH
Related in: MedlinePlus