Limits...
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.

Show MeSH

Related in: MedlinePlus

(A) EMSA conducted with increasing amounts of purified CPEB RRM1 or RRM1–RRM2 protein: 0, 1, 25, 50, 75, 100, 125, 150, 175 and 200 (mM) and radiolabelled U5A2U1 RNA (2 nM). Samples were fractionated in native gels and visualized by autoradiography. (B) ITC curves and affinity values obtained for the titration of CPEB4 RRM1–RRM2 with U5A2U1 (blue) and U5A1U2 (orange) RNAs. Data were acquired at 5°C in Tris buffer pH 7.0. The stoichiometry obtained was 0.6 and 0.7 respectively. The data were fitted using the independent model assuming a single binding site. (C) Protein samples were analyzed under semi-native conditions using SDS 12% PAGE. SDS was not present in the loading buffer and the samples were not boiled unless indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150798&req=5

Figure 4: (A) EMSA conducted with increasing amounts of purified CPEB RRM1 or RRM1–RRM2 protein: 0, 1, 25, 50, 75, 100, 125, 150, 175 and 200 (mM) and radiolabelled U5A2U1 RNA (2 nM). Samples were fractionated in native gels and visualized by autoradiography. (B) ITC curves and affinity values obtained for the titration of CPEB4 RRM1–RRM2 with U5A2U1 (blue) and U5A1U2 (orange) RNAs. Data were acquired at 5°C in Tris buffer pH 7.0. The stoichiometry obtained was 0.6 and 0.7 respectively. The data were fitted using the independent model assuming a single binding site. (C) Protein samples were analyzed under semi-native conditions using SDS 12% PAGE. SDS was not present in the loading buffer and the samples were not boiled unless indicated.

Mentions: In order to characterize the interaction of the RRM domains with short RNAs containing the CPE motifs, we have used several complementary techniques that allow us to detect and quantify the interactions. For this purpose, we performed EMSA as well as ITC binding assays using both RRM1 and RRM1–RRM2 and different RNA fragments. The EMSA experiments showed that RRM1–RRM2 tandem domain has a higher affinity for the CPE RNA when compared to the RRM1 alone (Figure 4A).


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) EMSA conducted with increasing amounts of purified CPEB RRM1 or RRM1–RRM2 protein: 0, 1, 25, 50, 75, 100, 125, 150, 175 and 200 (mM) and radiolabelled U5A2U1 RNA (2 nM). Samples were fractionated in native gels and visualized by autoradiography. (B) ITC curves and affinity values obtained for the titration of CPEB4 RRM1–RRM2 with U5A2U1 (blue) and U5A1U2 (orange) RNAs. Data were acquired at 5°C in Tris buffer pH 7.0. The stoichiometry obtained was 0.6 and 0.7 respectively. The data were fitted using the independent model assuming a single binding site. (C) Protein samples were analyzed under semi-native conditions using SDS 12% PAGE. SDS was not present in the loading buffer and the samples were not boiled unless indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150798&req=5

Figure 4: (A) EMSA conducted with increasing amounts of purified CPEB RRM1 or RRM1–RRM2 protein: 0, 1, 25, 50, 75, 100, 125, 150, 175 and 200 (mM) and radiolabelled U5A2U1 RNA (2 nM). Samples were fractionated in native gels and visualized by autoradiography. (B) ITC curves and affinity values obtained for the titration of CPEB4 RRM1–RRM2 with U5A2U1 (blue) and U5A1U2 (orange) RNAs. Data were acquired at 5°C in Tris buffer pH 7.0. The stoichiometry obtained was 0.6 and 0.7 respectively. The data were fitted using the independent model assuming a single binding site. (C) Protein samples were analyzed under semi-native conditions using SDS 12% PAGE. SDS was not present in the loading buffer and the samples were not boiled unless indicated.
Mentions: In order to characterize the interaction of the RRM domains with short RNAs containing the CPE motifs, we have used several complementary techniques that allow us to detect and quantify the interactions. For this purpose, we performed EMSA as well as ITC binding assays using both RRM1 and RRM1–RRM2 and different RNA fragments. The EMSA experiments showed that RRM1–RRM2 tandem domain has a higher affinity for the CPE RNA when compared to the RRM1 alone (Figure 4A).

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