To achieve the results highlighted below, a large number of protocols were developed/optimised for the effective use of the biochemical and biophysical techniques employed, either with the proteins alone or in complex with oligoA15 RNA. Extensive molecular biology and protein purification was performed: a set of 15 deletion and point mutants of LARP4 was generated and their expression and purification was optimised to get protein samples of high purity and significant quantity for the experiments needed. The group of techniques used include Nuclear Magnetic Resonance (NMR), Electrophoretic Mobility Shift Assays (EMSA), Circular Dichroism (CD), Microscale Thermophoresis (MST), Differential Scanning Fluorimetry (DSF), Isothermal Titration Calorimetry (ITC) and Size Exclusion Chromatography coupled to Multiangle Light scattering (SEC-MALLS) and pull downs.
The large number of experiments conducted has significantly furthered our understanding of how LARP4 binds its RNA partner. We revealed unexpected findings (listed below) that, given the high-profile novelty, focused the project more towards molecular recognition and less on cellular biology. A manuscript reported on this work is about to be submitted to a high impact journal.
The main achievements are:
• We have unveiled a completely novel way by which LARP4 recognises its RNA partner, using flexible regions that do not map to any previously identify RNA binding motif (Images 1 and 2).
• The RNA interaction site of LARP4 is a combination of secondary structure elements and disordered regions, which constitutes a breakthrough for the field of RNA-binding proteins and RNA metabolism.
• The La-module structure revealed that it is formed by two independently folded globular domains, the LaM and the RRM1, connected by a short linker (Image1). Although it was initially anticipated to be the main region for RNA binding, unexpectedly was found to play a minor role (Image 2).
• We have revealed an interesting and important interplay between protein-RNA and protein-protein interaction, in that LARP4 binding to oligoA is mutually exclusive with PABP (poly-A binding protein, another RNA binding protein central to life of cell and organisms).
