SummaryMicro-RNAs (miRNAs) are one class of endogenous tiny RNAs that play important regulatory roles in plant development and responses to external stimuli. To date, miRNAs have been cloned from higher plants such as Arabidopsis, rice and pumpkin, and there is limited information on their identity in lower plants including Bryophytes. Bryophytes are among the oldest groups of land plants among the earth's flora, and are important for our understanding of the transition to life on land. To identify miRNAs that might have played a role early in land plant evolution, we constructed a library of small RNAs from the juvenile gametophyte (protonema) of the moss Physcomitrella patens. Sequence analysis revealed five higher plant miRNA homologues, including three members of the miR319 family, previously shown to be involved in the regulation of leaf morphogenesis, and miR156, which has been suggested to regulate several members of the SQUAMOSA PROMOTER BINDING-LIKE (SPL) family in Arabidopsis. We have cloned PpSBP3, a moss SPL homologue that contains an miR156 complementary site, and demonstrated that its mRNA is cleaved within that site suggesting that it is an miR156 target in moss. Six additional candidate moss miRNAs were identified and shown to be expressed in the gametophyte, some of which were developmentally regulated or upregulated by auxin. Our observations suggest that miRNAs play important regulatory roles in mosses.

SummaryTrans-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 miRNAguided 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.

SummaryMicro-RNAs (miRNAs) are one class of small non-coding RNAs that have important regulatory roles in higher plants. Much less is known about their prevalence and function in lower land plants. Previously we cloned 100 non-structural small RNAs from the moss Physcomitrella patens but could annotate only 11 as miRNAs. To identify additional moss miRNAs among cloned small RNAs we have analyzed their genomic sequences for a characteristic miRNA precursor-like structure. This analysis revealed 19 new moss miRNAs that are predicted to be encoded by 22 putative foldbacks. Northern blot analysis confirmed the expression of 14 new miRNA representatives. Half of these were gametophore specific, the rest were detected at low levels in the protonema. We predicted 12 genes as targets of nine new miRNAs. Three of these show homology to transcription factors and the others appear to play roles in diverse physiological processes including light and cytokine signaling, which have not to date been shown to be regulated by a miRNA in flowering plants. Four target genes, which show homology to ATN1-like protein kinase, NAC transcription factors and a cytokinin receptor, have been validated by miRNA-mediated mRNA cleavage. In addition, our analysis revealed that seven small RNAs represent miRNA* and three represent intermediates of pre-miRNA processing, providing evidence for specific DICER-like cleavage steps during miRNA biogenesis in moss. Our findings suggest that miRNAs are common in mosses and set the stage for the elucidation of their varied biological functions.