The effect of RNA polymerase V on 24-nt siRNA accumulation depends on DNA methylation contexts and histone modifications in rice

Abstract

RNA-directed DNA methylation (RdDM) functions in de novo methylation in CG, CHG, and CHH contexts. Here, we performed map-based cloning of OsNRPE1, which encodes the largest subunit of RNA polymerase V (Pol V), a key regulator of gene silencing and reproductive development in rice. We found that rice Pol V is required for CHH methylation on RdDM loci by transcribing long noncoding RNAs. Pol V influences the accumulation of 24-nucleotide small interfering RNAs (24-nt siRNAs) in a locus-specific manner. Biosynthesis of 24-nt siRNAs on loci with high CHH methylation levels and low CG and CHG methylation levels tends to depend on Pol V. In contrast, low methylation levels in the CHH context and high methylation levels in CG and CHG contexts predisposes 24-nt siRNA accumulation to be independent of Pol V. H3K9me1 and H3K9me2 tend to be enriched on Pol V–independent 24-nt siRNA loci, whereas various active histone modifications are enriched on Pol V–dependent 24-nt siRNA loci. DNA methylation is required for 24-nt siRNAs biosynthesis on Pol V–dependent loci but not on Pol V–independent loci. Our results reveal the function of rice Pol V for long noncoding RNA production, DNA methylation, 24-nt siRNA accumulation, and reproductive development.

A null allele of the pol IV second subunit impacts stature and reproductive development in Oryza sativa

“…In comparison with wild type, nrpd2 mutants show approximately 69% reduction of 24-nt siRNAs (Figure 1c). This reduction is consistent with nrpd2 mutants in Arabidopsis (Zhang et al, 2007), and nr-pd1 mutants in rice (Debladis et al, 2020;Xu et al, 2020;Zheng et al, 2021). 21-and 22-nt sRNAs are elevated in nr-pd2 populations, most likely due to oversampling of these sizes following depletion of the dominant 24-nt size class.…”

“…Embryonic lethality was also reported for nrpd1 mutants; however, we observe homozygous mutant seeds arising at the expected ratios, indicating there is no lethality for homozygous mutant embryos. NRPE1 mutations in different rice strains result in different phenotypes (Zheng et al., 2021), suggesting that genomic differences between rice strains, particularly with respect to transposon insertion site, might explain the differences between nrpd1 and nrpd2 phenotypes we observed, although we cannot rule out the possibility of a secondary mutation linked to this T‐DNA insertion.…”

“…To compare directly the function of NRPD2 with NRPD1, we obtained sRNA sequencing data from rice nrpd1a nrpd1b double mutants ( nrpd1 , hereafter) (Zheng et al., 2021) and used the 300‐bp non‐overlapping windows to count sRNAs. There was a good correlation between wild‐type sRNA accumulation in leaf and seedling tissues (Figure 2a).…”

“…Mutation of NRPD1 causes a near‐complete loss of 24‐nt siRNAs, while disruption of NRPE1 causes only a small decrease in 24‐nt siRNA accumulation (Debladis et al., 2020; Gouil & Baulcombe, 2016; Grover et al., 2018; Erhard et al., 2009; Mosher et al., 2008; Wang et al., 2020; Xu et al., 2020; Zheng et al., 2021). However, because both Pol IV and Pol V are required for RdDM, CHH methylation is reduced when either largest subunit is eliminated (Gouil & Baulcombe, 2016; Stroud et al., 2013; Zheng et al., 2021). Although Arabidopsis nrpd1 or nrpe1 mutants develop normally, loss of NRPD1 or NRPE1 in many other species causes developmental defects (Gouil & Baulcombe, 2016; Grover et al., 2018; Hollick, 2010; Xu et al., 2020; Wang et al., 2020; Zhang et al., 2020; Zheng et al., 2021).…”

“…In the grass family, NRPE1 and NRPD2 duplicated, giving rise to two pairs of paralogous genes: NRPE1 and NRPF1 , and NRPD2 and NRPF2 respectively (Trujillo et al., 2018). Phylogenetic analysis suggests that NRPF1 and NRPF2 have new functions, and genetic analysis determined that NRPE1 and NRPF1 are non‐redundant (Trujillo et al., 2018; Zheng et al., 2021).…”