Mechanisms of microRNA Processing in Plants

NICOLÁS G BOLOGNA; ARNALDO L SCHAPIRE; JIXIAN ZHAI; BLAKE MEYERS; JAVIER F PALATNIK

Vienna

Workshop; Plant RNA Workshop; 2012

MicroRNAs (miRNAs) are transcribed as larger transcripts that contain fold-back structures that are processed by RNAse III complexes. In both plants and animals, the precursor contains spatial clues that determine the position of the miRNA along its sequence. However, at a difference with their animal counterparts, plant precursors are very heterogeneous in size and shape. They are processed completely in the nucleus by a complex formed by DICER-LIKE1 (DCL1) and accessory proteins such as HYL1 and SERRATE. A random mutagenesis approach on miR172 precursor revealed that a 15 nucleotide lower stem below the miRNA is essential for its processing. This structural determinant present in many, but not all plant miRNAs, is recognized by the processing machinery to produce a first cleavage at the base of the precursor, while a second cut below the loop releases the miRNA. In contrast, we found that the biogenesis of miR319 and miR319 proceeds through a loop-to-base direction. DCL1 produces first a cleavage at the loop of the precursor and continues towards the base of the precursor, while the mature miRNAs are finally release between the third and fourth cleavage sites. This non-canonical processing mechanism highlights the plasticity of the plant miRNA pathway. To bring insights into the biogenesis of miRNAs from a genome-wide perspective in Arabidopsis thaliana, we designed a strategy to detect precursor intermediates using high-throughput sequencing. Using this strategy we were able to map the cleavage sites for most Arabidopsis miRNA precursors. We found a good correlation between the cuts in the precursors and the miRNA profile obtained by deep-sequencing small RNAs. A genome-wide view of miRNA processing in Arabidopsis will be presented.