Research Grants

Award Year 2006

BOISBOUVIER Jérôme
PALATNIK Javier
RNA Shape Recognition And Structure In MicroRNA Processing

MicroRNAs (miRNAs) are small RNAs of 20-23 nt that recognize partially complementary sites in target mRNAs and guide them to cleavage or translational arrest. The genomes of plants and animals encode hundreds of miRNAs that regulate a substantial fraction of their genes. MiRNAs are transcribed as larger precursors that form fold-back structures and are recognized by RNAse III enzymes, which degrade the precursor to release the mature miRNA. But to date, the exact miRNA sequence derived from a given precursor remains unpredictable. The precursor supposedly contains spatial determinants that indicate the position of the miRNA along its sequence. Rather surprisingly, currently no miRNA precursor structure has been experimentally determined. We propose to contribute to the understanding of RNA structure and the recognition of RNA conformation during miRNA processing in plants. First, we will solve the spatial structure of an Arabidopsis thaliana miRNA precursor by solution NMR. Second, using mutagenesis we will isolate precursors with processing defects. Structural and biochemical data obtained in vitro will be integrated with genetic information and in vivo data. This multidisciplinary approach will be used to map key nucleotides and base pairs required for correct processing. Once we have determined RNA structural and functional requirements for miRNA processing, we will study the RNA-protein interactions during this process. Protein domains from components of the processing machinery will be positioned on a miRNA precursor based on the NMR detection of intermolecular long-range restraints in the whole processing precursor complex. Special attention will be paid to the determination of crucial intermolecular interactions responsible for the specificity of the miRNA sequence cleaved from the larger precursor. The integration of the obtained results will be used to model miRNA processing. Finally, in a recursive and interactive manner, rational mutations will be designed to test, adjust and validate the proposed model.