Digestive organs at 3 days development

Andres Ramos group project:

Methods to study protein-RNA interactions

We are working to expand our capability to analyse protein-RNA interaction at the molecular level. We focus on two main areas:

  1. Molecular determinants of sequence specificity. We need tools to analyse the determinants of sequence specificity in vitro and bridge the gap between structural information and in vivo high through-put data. In collaboration with the group of Ian Taylor (Molecular Structure), we have developed a Scaffold Independent Analysis method (Figure 5) that provides a complete map of the nucleic acid sequence preference of a protein domain (i.e. the nucleobase ranking for each of the binding positions)(Beuth, B et al, 2007, García-Mayoral et al, 2008). We plan to expand the method to analyse more complex systems and to increase the through-put of the analysis.
  2. Long range constraints in structure determination of protein-RNA complexes. The study of the interfaces of the large and flexible protein-RNA complexes regulating mRNA metabolism is difficult, because of flexibility at the interface and of the complexity of these systems. We are developing cross-saturation experiments (Ramos et al, 2000) to define the protein and nucleic acid surfaces responsible for complexation and paramagnetic probes (Ramos et al, 1998) to provide long-range PRE restraints for structure calculation.


    3. Flow chart of a Sequence Independent Analysis experiment on an RNA binding domain

    Flow chart of a Sequence Independent Analysis experiment on an RNA binding domain

    Click image to view at full-size

    Design of appropriate oligo pools, NMR titration the protein domain with the pools and measure of chemical shift changes, normalisation and averaging of the results, nucleobase ranking and validation of the data.

    Selected publications

    • García-Mayoral, MF; Díaz-Moreno, I; Hollingworth, D and Ramos, A (2008)
      The sequence selectivity of KSRP explains its flexibility in the recognition of the RNA targets.
      Nucleic Acids Research 36, 5290-5296 PubMed abstract
    • Beuth, B; García-Mayoral, MF; Taylor, IA and Ramos, A (2007)
      Scaffold-independent analysis of RNA-protein interactions: The Nova-1 KH3-RNA complex.
      Journal of the American Chemical Society 129, 10205-10210 PubMed abstract

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