PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis

Abstract

DNA-dependent RNA polymerase (Pol)IV in Arabidopsis exists in two isoforms (PolIVa and PolIVb), with NRPD1a and NRPD1b as their respective largest subunits. Both isoforms are implicated in production and activity of siRNAs and in RNA-directed DNA methylation (RdDM). Deep sequence analysis of siRNAs in WT Arabidopsis flowers and in nrpd1a and nrpd1b mutants identified >4,200 loci producing siRNAs in a PolIV-dependent manner, with PolIVb reinforcing siRNA production by PolIVa. Transposable element identity and pericentromeric localization are both features that predispose a locus for siRNA production via PolIV proteins and determine the extent to which siRNA production relies on PolIVb. Detailed analysis of DNA methylation at PolIV-dependent loci revealed unexpected deviations from the previously noted association of PolIVb-dependent siRNA production and RdDM. Notably, PolIVb functions independently in DNA methylation and siRNA generation. Additionally, we have uncovered siRNA-directed loss of DNA methylation, a process requiring both PolIV isoforms. From these findings, we infer that the role of PolIVb in siRNA production is secondary to a role in chromatin modification and is influenced by chromatin context.

Genome-Wide Characterization of Maize Small RNA Loci and Their Regulation in the required to maintain repression6-1 (rmr6-1) Mutant and Long-Term Abiotic Stresses

“…Once loaded onto ARGONAUTE 4, these siRNAs are thought to target nascent, chromatin-associated noncoding RNAs transcribed by RNA Pol V. Successful targeting correlates with the deposition of de novo DNA methylation and other repressive epigenetic marks at the target loci, inducing TGS (for review, see Matzke and Mosher, 2014). Pol IV-dependent siRNAs are often produced from transposable elements (TEs), TE-like sequences, and other repeats (Zhang et al, 2007;Mosher et al, 2008). They contribute to the reinforcement of TE silencing (Slotkin et al, 2005;Marí-Ordóñez et al, 2013;Nuthikattu et al, 2013) and in some cases are also essential to control the expression of proteincoding genes in cis or in trans (Liu et al, 2004;Kinoshita et al, 2007;McCue et al, 2013).…”

Epigenetics: heterochromatin meets RNAi

“…Deep sequencing of small RNAs from NRPD1a and NRPD1b mutants revealed transposons and centromeres as key sources of NRPD1a-dependent siRNAs [40]. Surprisingly, the effect of mutant NRPD1b on DNA methylation is separable from its effect on siRNA accumulation, indicating more complex mechanisms of pol IV action than previously anticipated.…”

“…The phenotypes of mutations in NRPD1a and NRPD1b differ, with that of NRPD1a being more severe and affecting a larger number of siRNA clusters [106]. Therefore NRPD1b, which only affects siRNA from a subset of the NRPD1a-dependent clusters, has been supposed to act downstream [40,106]. NRPD1a and b differ mainly in their C-terminal domains.…”

“…Endogenous short interfering RNA (siRNA) originates from repetitive sequences, such as centromeric repeats, transposable elements or rDNA. Several different siRNA molecules along the length of the double-strand are detectable [39] and make up a considerable fraction of the short RNAs that have been cloned [29,[40][41][42][43][44]. The length of the siRNA varies between species, usually in between 19-22 nucleotides up to 26 nucleotides, and depends on the specificity of the enzymes that produce them.…”

The Cornucopia of Small RNAs in Plant Genomes

“…In the siRNA processing center, the largest subunit of Pol IVb joins the AGO4-containing RISC complex and guides DNA methylation and heterochromatic histone (H3K9) modifications at the endogenous repeats [55][56][57][58][59][60][61]. A recent study has uncovered that, in several loci of Arabidopsis, Pol IVb's role as the effector of RNA silencing is independent of its function in siRNA biogenesis, and the study proposed that some epigenetic marks of chromatin adjacent to the Pol IVbtargeted region could influence the ability of Pol IVb-guided DNA methylation [62]. Although there is evidence showing that heterochromatic siRNAs can trigger epigenetic effects at the target loci, a recent study revealed that some endogenous rice genes, including OsRac, are rarely transcriptionally silenced by promoter-targeted siRNAs, but these genes could be post-transcriptionally suppressed by RNA interference (RNAi) [63].…”