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

Abstract

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.

Revisiting Criteria for Plant MicroRNA Annotation in the Era of Big Data

“…However, homologs of key genes known to be responsible for hcsiRNA biogenesis and function in angiosperms clearly exist in diverse plant lineages (Zong et al, 2009;Banks et al, 2011;Huang et al, 2015;Wang and Ma, 2015;You et al, 2017). Reverse genetic analyses of these homologs, coupled with sRNA-seq analyses of the mutants, has shown that hc-siRNAs exist in the moss P. patens (Cho et al, 2008;Coruh et al, 2015), which implies that the pathway was most likely present in the last common ancestor of all land plants. Whether hc-siRNAs have been specifically lost in the conifers and/or ferns remains an open question, but the presence of high levels of 24 nt RNAs in specific tissues of the conifers Norway spruce (Picea abies) (Nystedt et al, 2013) and Japanese larch (Larix leptolepis) (Zhang et al, 2013) suggests that this may not be the case.…”

Helitrons , the Eukaryotic Rolling-circle Transposable Elements

“…Small RNAs homologous to Helitron sequences have been described from maize (67), A. thaliana (134), Phytophthora species, (187,188) and Physcosmitrella patens (189). Generally it is surmised that one kind of small RNA (small interfering (siRNA)) regulate TE expression.…”

Sliced microRNA targets and precise loop-first processing of MIR319 hairpins revealed by analysis of the Physcomitrella patens degradome

“…wild-type 10-d-old protonemata (NCBI GEO GSM313212) (Cho et al 2008). This had the effect of removing from consideration MIRNAs which were not highly expressed in the tissue from which the degradome sample was taken.…”

“…Wild-type 10-d-old Physcomitrella patens (Gransden) protonemata were cultured, harvested, and total RNA extracted as previously described (Cho et al 2008). Approximately 200 ng of polyA-enriched RNA were directly ligated to a chimeric DNA/ RNA adapter containing a 39 EcoP15I site using T4 RNA Ligase 1 (New England Biolabs).…”