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miRNASTRESS: structural and functional study of the RBM38-controlled mechanism of mRNA stabilization

Final Report Summary - MIRNASTRESS (miRNASTRESS: structural and functional study of the RBM38-controlled mechanism of mRNA stabilization.)

A fine balance between cell death and cell proliferation is necessary for the physiological development of an organism and loss of this balance is correlated to the insurgence of cancer. The pathways regulating cell death and proliferation converge on a few key genes. The expression of these genes is up or down-regulated in response to stress and other environmental inputs and needs to be tightly regulated.
This project investigates the regulation of key apoptosis and cellular proliferation genes p21, c-myb and c-myc in response to changes in cellular conditions, such as stress. This regulation takes place at multiple steps of the metabolism of the mRNA codified by these genes, and we have investigated both regulation of mRNA synthesis and regulation of mRNA translation and decay. Our initial focus has been on understanding how the nucleic acid binding protein RBM38 recognises its RNA targets and mediates p53 regulation of p21 and c-myb translation. In the second part of the project we have looked at the regulation of the synthesis of p21 mRNA by the protein FIR. Mis-function of these two regulatory mechanisms is closely associated with specific cancers and the insight in the selectivity and the mechanism of regulation provided by this work is important both for the understanding of the system and for the design of future therapies.

Upon cellular stress the RNA binding protein RBM38 competes with microRNAs (miRNAs) to stabilize mRNAs of proteins required by p53 to regulate cell proliferation and apoptosis. Our aim was to investigate how RBM38 selects specific targets and inhibits the action of the target miRNAs. We have used Scaffold Independent Analysis (SIA) to identify the overall RNA sequence preference of RBM38 (which we found to be U/G) and NMR and other biophysical techniques to map the exact location of the binding sites within the target miRNA sequences. We show these sites display a partial overlap with some of the mRNA sequences bound by the miRNAs. Then we have used UV melting and imino proton NMR experiments to show that the miRNA-mRNA duplex is indeed destabilized by the presence of the protein. Finally, we have characterized the protein-RNA interaction and showed that the RNA binds on the canonical nucleic acid binding surface of the RRM domain. However, the interaction between RBM38 and the RNA targets is a dynamic one confirming that other factors are likely to contribute to the binding.
Overall, our data confirm that the action of RBM38 is mediated by a specific recognition of U/G target sequences and by a direct destabilization/competition with the miRNA-mRNA interaction. We expect this description of the specificity and RNA binding properties of the RBM38 protein will be useful to manipulate its action on p21 and other targets.

FUSE-mediated regulation of the p21 and c-myc genes relies on the binding of an activator (the FUSE Binding Protein, or FBP) and a repressor (the FBP Interacting Repressor, or FIR) protein to a ssDNA element upstream of the target genes (Far Upstream Regulatory Element, FUSE). FUSE-mediated mis-regulation of p21 and c-myc is linked respectively to hepatic cancer and kidney and gut cancer.
We have defined the FIR cognate sequence on the FUSE DNA and investigated the structural details of the interaction. We are currently refining the three-dimensional high-resolution structure of FIR in complex with a DNA target using NMR spectroscopy, while designing mutants with modified DNA-binding properties (affinity and specificity). These mutants will be used to probe if, for example, a moderate increase in the affinity between FIR and the DNA would lead to a decrease in the peak in the concentration of Myc or p21 in the cell – which would confirm the potential of FIR as anticancer target.

Our results on how two nucleic acid binding proteins regulate specifically the synthesis and translation of the p21 mRNA provide the molecular basis to design strategies to treat p21-dependent cancer.