Functional Dissection of the Pol V Largest Subunit CTD in RNA-Directed DNA Methylation

Abstract

Summary Plant multisubunit RNA Polymerase V transcription recruits Argonaute-siRNA complexes that specify sites of RNA-directed DNA methylation (RdDM) for gene silencing. Pol V’s largest subunit, NRPE1, evolved from the largest subunit of Pol II but has a distinctive carboxyl-terminal domain (CTD). We show that the Pol V CTD is dispensable for catalytic activity in vitro, yet essential in vivo. One CTD subdomain (DeCL) is required for Pol V function at virtually all loci. Other CTD subdomains have locus-specific effects. In a yeast two-hybrid screen, the 3′–>5′ exoribonuclease, RRP6L1 was identified as an interactor with the DeCL and glutamine-serine-rich (QS) subdomains located downstream from an Argonaute-binding subdomain. Experimental evidence indicates that RRP6L1 trims the 3′ ends of Pol V transcripts sliced by ARGONAUTE 4 (AGO4), suggesting a model whereby the CTD enables the spatial and temporal coordination of AGO4 and RRP6L1 RNA processing activities.

Common alleles of CMT2 and NRPE1 are major determinants of CHH methylation variation in Arabidopsis thaliana

“…Second, the use of more fine-grained phenotypes allowed to refine previous associations, identifying candidate causal polymorphisms in both CMT2 and NRPE1 (Fig 2). Furthermore, by comparing the genome-wide mCHH pattern with published data for loss-of-function mutations [13,19], we were able to establish the causal involvement of these genes (Figs 3 and 4).…”

“…https://doi.org/10.1371/journal.pgen.1008492.g003 NRPE1', by contrast, is clearly less specific, and showed strong correlations with loss-offunction phenotypes of nine genes in the RdDM pathway (including, of course, NRPE1 itself). However, since none of these genes, nor any other plausible candidate, is located near NRPE1 (S5 Fig), it seems reasonable to assume that the non-synonymous polymorphisms in this gene, particularly in RNA polymerase domain, cause a phenotype similar to knocking out NRPE1 [19], rather than by somehow regulating an unknown member of the RdDM pathway (S5 and S6 Figs). The relative lack of specificity of NRPE1 can also be seen from the comparison of natural alleles and knock-out mutations.…”

Common alleles ofCMT2andNRPE1are major determinants ofde novoDNA methylation variation inArabidopsis thaliana

“…2). Furthermore, by comparing the genome-wide mCHH pattern with published data for loss-of-function mutations [13, 19], we were able to establish the causal involvement of these genes (Figs 3-4).…”

“…NRPE1’ , by contrast, is clearly less specific, and showed strong correlations with loss-of-function phenotypes of nine genes in the RdDM pathway (including, of course, NRPE1 itself). However, since none of these genes, nor any other plausible candidate, is located near NRPE1 (S5 Fig), it seems reasonable to assume that the non-synonymous polymorphisms in this gene, particularly in RNA polymerase domain, cause a phenotype similar to knocking out NRPE1 [19], rather than by somehow regulating an unknown member of the RdDM pathway (S5-6 Figs). The relative lack of specificity of NRPE1 can also be seen from the comparison of natural alleles and knock-out mutations.…”

“…DML induced by mutants was calculated by the same way between wild-type and 86 loss-of-function mutants (Fig. 4; GSE39901; [13]) and nrpe1 mutants (GSE93558 [19]). Spearman’s correlation coefficients were calculated between DML for natural alleles, and mutants and empirical p -values were estimated using permutation test with 1500 randomly picked up SNPs along to genome (S4 Fig).…”