Non-Canonical Processing of Arabidopsis pri-miR319a/b/c Generates Additional microRNAs to Target One RAP2.12 mRNA Isoform

Lukasz Sobkowiak, Wojciech Karlowski, Artur Jarmolowski, Zofia Szweykowska-Kulinska

Arabidopsis miR319a/b/c primary transcripts are unusual due to the presence of a long stem and loop structure containing functional miR319a/b/c molecules. In our experiments carried out using high throughput sequencing (HTS), we have shown that additional microRNAs (miRNAs), miR319a.2/b.2/c.2 are generated from the upper part of the same hairpin structure. We have also found cognate miRNAa.2*/b.2*/c.2* to be present in the HTS results with a considerably lower number of reads. Northern hybridization revealed that miR319b.2 is mainly expressed in 35-day-old plant rosette leaves, as well as in stem and inflorescences of 42- and 53-day-old plants. Moreover, it carries multiple signatures of a functional miRNA, including as follows: (i) its biogenesis is HYL1-dependent; (ii) it is incorporated in a substantial amount into RISC complexes containing AGO1, AGO2, or AGO4 protein; (iii) 24 nt-long species of miR319b.2 have been found in inflorescences to be more abundant than 21 nt miR319b.2 species; (iv) it is present in various ratios to miR319b during plant development, which suggests the existence of a regulatory mechanism responsible for its biogenesis/processing; (v) there is an observed cross-species conservation of the miR319a/b/c stem nucleotide sequence extending beyond mature miRNA region; and (vi) all evidence suggests that intron-containing RAP2.12 mRNA isoform is the target for miR319b.2. All these features prompt us to claim miR319b.2 as a functional miRNA molecule.