MECHANISMS OF microRNA PROCESSING IN PLANTS.

BOLOGNA, N., MATEOS, J., RASIA, R., BOISBOUVIER J., PALATNIK J.

Bariloche, Argentina

Congreso; ICGEB meeting,; 2007

ICGEB

MicroRNAs (miRNAs) are small RNAs of ~21 nt that recognize partially complementary sites in target mRNAs and guide them to cleavage or translational arrest. They are transcribed as larger transcripts that contain fold-back structures that are processed by RNAse III complexes. In animals, the nuclear localized DROSHA assisted by DGCR8 cleaves the base of the stem-loop, which is then exported to the cytoplasm where a second cleavage by DICER releases the mature miRNA. Plant miRNAs are completely processed in the nucleus by DICER-LIKE1 (DCL1) assisted by HYL1. 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 animals, plant miRNA precursors are heterogeneous in size and shape, and the processing mechanisms still remain largely unknown. We are studying miRNA processing in plants using two main approaches. On one hand, we are performing in vitro studies to analyze the interaction between the RNA binding domains of HYL1 and DCL1 and miRNA precursors. On the other hand, we are analyzing the sequence requirements for miRNA processing in Arabidopsis thaliana. We have focused on miR172 and miR319 that regulate AP2 and TCP transcription factors respectively. Overexpression of miR172 precursor causes an early transition from vegetative to reproductive phase, while miR319 causes an increased cell proliferation and changes in leaf morphogenesis. Analysis of the processing intermediates of these miRNAs revealed that the miR319 precursor requires four sequential cuts to release the mature miRNA, instead of the usual two. We have performed mutagenesis on both precursors and selected mutations that interfere with the overexpression phenotype observed in Arabidopsis plants. The effects of these sequence changes on miRNA biogenesis in plants are currently under analysis.Arabidopsis thaliana. We have focused on miR172 and miR319 that regulate AP2 and TCP transcription factors respectively. Overexpression of miR172 precursor causes an early transition from vegetative to reproductive phase, while miR319 causes an increased cell proliferation and changes in leaf morphogenesis. Analysis of the processing intermediates of these miRNAs revealed that the miR319 precursor requires four sequential cuts to release the mature miRNA, instead of the usual two. We have performed mutagenesis on both precursors and selected mutations that interfere with the overexpression phenotype observed in Arabidopsis plants. The effects of these sequence changes on miRNA biogenesis in plants are currently under analysis.