Physcomitrella patens DCL3 Is Required for 22–24 nt siRNA Accumulation, Suppression of Retrotransposon-Derived Transcripts, and Normal Development

Endogenous 24 nt short interfering RNAs (siRNAs), derived mostly from intergenic and repetitive genomic regions, constitute a major class of endogenous small RNAs in flowering plants. Accumulation of Arabidopsis thaliana 24 nt siRNAs requires the Dicer family member DCL3, and clear homologs of DCL3 exist in both flowering and non-flowering plants. However, the absence of a conspicuous 24 nt peak in the total RNA populations of several non-flowering plants has raised the question of whether this class of siRNAs might, in contrast to the ancient 21 nt microRNAs (miRNAs) and 21–22 nt trans-acting siRNAs (tasiRNAs), be an angiosperm-specific innovation. Analysis of non-miRNA, non-tasiRNA hotspots of small RNA production within the genome of the moss Physcomitrella patens revealed multiple loci that consistently produced a mixture of 21–24 nt siRNAs with a peak at 23 nt. These Pp23SR loci were significantly enriched in transposon content, depleted in overlap with annotated genes, and typified by dense concentrations of the 5-methyl cytosine (5 mC) DNA modification. Deep sequencing of small RNAs from two independent Ppdcl3 mutants showed that the P. patens DCL3 homolog is required for the accumulation of 22–24 nt siRNAs, but not 21 nt siRNAs, at Pp23SR loci. The 21 nt component of Pp23SR-derived siRNAs was also unaffected by a mutation in the RNA-dependent RNA polymerase mutant Pprdr6. Transcriptome-wide, Ppdcl3 mutants failed to accumulate 22–24 nt small RNAs from repetitive regions while transcripts from two abundant families of long terminal repeat (LTR) retrotransposon-associated reverse transcriptases were up-regulated. Ppdcl3 mutants also displayed an acceleration of leafy gametophore production, suggesting that repetitive siRNAs may play a role in the development of P. patens. We conclude that intergenic/repeat-derived siRNAs are indeed a broadly conserved, distinct class of small regulatory RNAs within land plants

Bioinformatic prediction and experimental validation of a microRNA-directed tandem trans-acting siRNA cascade in Arabidopsis

Small RNAs play pivotal roles in regulating gene expression in higher eukaryotes. Among them, trans-acting siRNAs (ta-siRNAs) are a class of small RNAs that regulate plant development. The biogenesis of ta-siRNA depends on microRNA-targeted cleavage followed by the DCL4-mediated production of small RNAs phased in 21-nt increments relative to the cleavage site on both strands. To find TAS genes, we have used these characteristics to develop the first computational algorithm that allows for a comprehensive search and statistical evaluation of putative TAS genes from any given small RNA database. A search in Arabidopsis small RNA massively parallel signature sequencing (MPSS) databases with this algorithm revealed both known and previously unknown ta-siRNA-producing loci. We experimentally validated the biogenesis of ta-siRNAs from two PPR genes and the trans-acting activity of one of the ta-siRNAs. The production of ta-siRNAs from the identified PPR genes was directed by the cleavage of a TAS2-derived ta-siRNA instead of by microRNAs as was reported previously for TAS1a, -b, -c, TAS2, and TAS3 genes. Our results indicate the existence of a small RNA regulatory cascade initiated by miR173-directed cleavage and followed by the consecutive production of ta-siRNAs from two TAS genes

Towards annotating the plant epigenome: the Arabidopsis thaliana small RNA locus map

Based on 98 public and internal small RNA high throughput sequencing libraries, we mapped small RNAs to the genome of the model organism Arabidopsis thaliana and defined loci based on their expression using an empirical Bayesian approach. The resulting loci were subsequently classified based on their genetic and epigenetic context as well as their expression properties. We present the results of this classification, which broadly conforms to previously reported divisions between transcriptional and post-transcriptional gene silencing small RNAs, and to PolIV and PolV dependencies. However, we are able to demonstrate the existence of further subdivisions in the small RNA population of functional significance. Moreover, we present a framework for similar analyses of small RNA populations in all species

AGO1-miR173 complex initiates phased siRNA formation in plants

MicroRNA (miRNA)-guided cleavage initiates entry of primary transcripts into the transacting siRNA (tasiRNA) biogenesis pathway involving RNA-DEPENDENT RNA POLYMERASE6, DICER-LIKE4, and SUPPRESSOR OF GENE SILENCING3. Arabidopsis thaliana TAS1 and TAS2 families yield tasiRNA that form through miR173-guided initiation–cleavage of primary transcripts and target several transcripts encoding pentatricopeptide repeat proteins and proteins of unknown function. Here, the TAS1c locus was modified to produce synthetic (syn) tasiRNA to target an endogenous transcript encoding PHYTOENE DESATURASE and used to analyze the role of miR173 in routing of transcripts through the tasiRNA pathway. miR173 was unique from other miRNAs in its ability to initiate TAS1c-based syn-tasiRNA formation. A single miR173 target site was sufficient to route non-TAS transcripts into the pathway to yield phased siRNA. We also show that miR173 functions in association with ARGONAUTE 1 (AGO1) during TAS1 and TAS2 tasiRNA formation, and we provide data indicating that the miR173–AGO1 complex possesses unique functionality that many other miRNA–AGO1 complexes lack

Evidence for widespread exonic small RNAs in the glaucophyte alga Cyanophora paradoxa

RNAi (RNA interference) relies on the production of small RNAs (sRNAs) from double-stranded RNA and comprises a major pathway in eukaryotes to restrict the propagation of selfish genetic elements. Amplification of the initial RNAi signal by generation of multiple secondary sRNAs from a targeted mRNA is catalyzed by RNA-dependent RNA polymerases (RdRPs). This phenomenon is known as transitivity and is particularly important in plants to limit the spread of viruses. Here we describe, using a genome-wide approach, the distribution of sRNAs in the glaucophyte alga Cyanophora paradoxa. C. paradoxa is a member of the supergroup Plantae (also known as Archaeplastida) that includes red algae, green algae, and plants. The ancient (>1 billion years ago) split of glaucophytes within Plantae suggests that C. paradoxa may be a useful model to learn about the early evolution of RNAi in the supergroup that ultimately gave rise to plants. Using next-generation sequencing and bioinformatic analyses we find that sRNAs in C. paradoxa are preferentially associated with mRNAs, including a large number of transcripts that encode proteins arising from different functional categories. This pattern of exonic sRNAs appears to be a general trend that affects a large fraction of mRNAs in the cell. In several cases we observe that sRNAs have a bias for a specific strand of the mRNA, including many instances of antisense predominance. The genome of C. paradoxa encodes four sequences that are homologous to RdRPs in Arabidopsis thaliana. We discuss the possibility that exonic sRNAs in the glaucophyte may be secondarily derived from mRNAs by the action of RdRPs. If this hypothesis is confirmed, then transitivity may have had an ancient origin in Plantae