Identification of trans-acting siRNAs in moss and an RNA-dependent RNA polymerase required for their biogenesis

Abstract

Trans-acting small interfering RNAs (tasiRNAs) are a class of higher-plant endogenous siRNAs that, like miRNAs, direct the cleavage of non-identical transcripts. tasiRNAs derive from non-coding transcripts (TAS) that are converted into dsRNA by a RNA-dependent RNA polymerase (RDR6), following their initial miRNA-guided cleavage. The dsRNA is then processed by a dicer-like enzyme 4 into phased 21-nucleotide siRNAs. To date, tasiRNAs have been identified only in Arabidopsis, and their identity and function in other land plants are unknown. Here, a set of endogenous small RNAs that correspond in a phased manner to a non-coding transcript (contig13502) were identified in the moss Pyscomitrella patens. Northern analysis suggests that contig13502-derived small RNAs are expressed in the juvenile gametophyte. In addition, miR390-guided cleavage of contig13502 at two sites flanking the small RNAs cluster was validated by 5' RACE. These cleavages are predicted to provide defined termini for the production of phased siRNAs. To elucidate the biogenesis of identified siRNAs, we cloned and generated knock-out mutants for an RDR6 moss homologue (PpRDR6). These mutants exhibited an accelerated transition from juvenile to mature gametophyte. In addition, RNA blots demonstrated that they lacked contig13502-derived siRNAs, suggesting that PpRDR6 is required for siRNA biogenesis. A target gene, which showed homology to an AP2/EREBP transcription factor, for one phased siRNA, was validated, corroborating its identity as a trans-acting siRNA. Taken together, our data indicate that contig13502 is a novel TAS locus and suggest a role for derived tasiRNAs in the regulation of gene expression in moss.