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Structural and functional studies of LARP4, a new RNA binding protein involved in mRNA stabilisation and cell migration

Periodic Reporting for period 1 - LARP4MOT (Structural and functional studies of LARP4, a new RNA binding protein involved in mRNA stabilisation and cell migration)

Reporting period: 2016-09-01 to 2018-08-31

In every single cell of our bodies, RNA (a chemical variant of DNA) provides the messenger that carries the DNA code information of the nucleus into the cytoplasm, where the proteins are made. However, it turns out that RNA has many other roles and the interplay between RNA-binding proteins and RNA molecules is now known to be central to the life of all cells and organisms. Nonetheless the roles of most RNA-binding proteins remain unknown. LARP4 is a newly discovered RNA-binding protein that is needed for cells to move efficiently. Since anomalous cell migration contributes to the development of human diseases including cancer, chronic inflammation and atherosclerosis, our work on elucidating how LARP4 functions in the cell will advance our understanding of key cellular mechanisms relating to RNA biology. The aim of this project is to understand exactly how LARP4 binds to RNA at the molecular level and how this impacts on cell morphology and migration, especially in the context of cancer cell biology.
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).
The exciting and novel outcomes of this project will have a transformative effect in the field of the La-related proteins (LARPs), and RNA binding biology in general. LARPs are potential candidates for cancer therapy and autoimmunity.
The work performed in the framework of this project is primarily fundamental research, and the biggest impact of the results will be on influencing the research direction of other scientists in academia, biotech and industry working on human diseases that affect a large percentage of the EU population and involve cell migration, cancer, chronic inflammation and heart disease.
Schematic representation of the RNA binding site of LARP4 protein
Model of LARP4 interaction to RNA