Primary sequence relevance in plant microRNA processing

MATEOS, JULIETA; CHOROSTECKI, UCIEL; SCHAPIRE, ARNALDO L; PALATNIK, JAVIER; DRUSIN, SALVADOR; MORO, BELEN; MORENO, DIEGO; ROJAS, ARANTXA M. L.; BOLOGNA, NICOLAS; BRESSO, EDGARDO; RASIA, RODOLFO

Kraków

Congreso; 24th Annual Meeting of the RNA Society; 2019

RNA Society

MicroRNAs (miRNAs) are small RNAs of 20-22 nucleotides present in plants and animals. They are coded within the genome and play a crucial role as post-transcriptional gene regulators. For the model plant, Arabidopsis thaliana (Arabidopsis), are 326 precursors and 428 mature sequences. These miRNAs regulate genes involved in development, hormone signaling and stress response. These small RNAs are generated from longer precursors with fold-back structures with the miRNA located in one of their arms. In plants, miRNA precursors are processed in the nuclei by a complex harboring the protein DICER-LIKE1 (DCL1). This complex recognizes structural determinants in the precursors to produce all the cuts and release the mature miRNA. To gain insights into MIRNA processing in plants, we performed a random mutagenesis of the MIR172a precursor of Arabidopsis with an emphasis in finding mutants with higher activity. miR172 regulates flowering time and flower patterning. Overexpression of miR172 produces early flowering plants with flower defects, resembling the ap2 phenotype. The strength of these phenotypes can therefore be used to assess processing efficiency of the mutant precursor. Through this approach, we identified hot spot positions for MIRNA processing. Interestingly, most of them were located at DCL1 cleavage sites. Small changes in these regions had a strong effect on the biogenesis of the small RNA. This allows us to define the cleavage sites architecture of Arabidopsis MIRNA precursors. Moreover, we show that the spatial configuration is usually conserved between angiosperms. Furthermore, we recovered a mutant precursor with a single change that enhance the accumulation of the miRNA by several folds. This demonstrates that even simple changes in the DCL1 cleavage site architecture can strongly modulate the biogenesis of miRNAs in plants. The results obtained provide new insights into the molecular basis of miRNA biogenesis in plants.