FUNCTIONAL STUDIES ON PLANT MICRORNA PRECURSORS.

MATEOS, J.L., BOLOGNA, N. AND PALATNIK, J.F.

Berlin, Alemania

Congreso; Annual Meeting of the RNA Society; 2008

RNA Society

MicroRNAs (miRNAs) are small, noncoding RNAs ~21 nt in length that derive from the specific cleavage of short hairpin precursors. Their functional role in gene regulation has been extensively shown in multicellular organisms. The main feature that distinguishes miRNAs from other small RNAs is their unique biogenesis. MiRNA biogenesis has been described rather extensively in animals. First, miRNA primary transcripts (pri-miRNAs) are cleaved in the nucleus into miRNA precursors (pre-miRNAs) by an RNase III-like enzyme called Drosha. After this initial processing, the pre-miRNAs are exported to the cytoplasm by the function of exportin-5 and are cleaved there to generate mature miRNAs by another RNase III-like enzyme called Dicer. At a difference, plant miRNAs are completely processed in the nucleus by DICER-like 1, and the mechanisms of plant miRNA biogenesis remains poorly understood. There are hundreds of different miRNA precursors encoded in animal and plant genomes, yet they do not seem to share any common sequence motifs. Interestingly, the size and shape of plant miRNA precursors are much more variable than their animal counterparts.   Here, we focused on the sequence requirements for plant miRNAs to have a precise processing in vivo. We performed site directed mutagenesis on multiple regions of selected plant miRNA precursors. The mutant precursors were overexpressed in Arabidopsis thaliana and scored for their ability to induce developmental defects usually caused by high miRNA levels. The transgenic plants expressing the different modified precursors were also analyzed biochemically. We also mapped precursor intermediates of processing showing that for this step Dicer-like-1 activity is strand specific. The results obtained allowed us to map specific precursor regions necessary for a proper processing of plant miRNA precursors.