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

RNA-directed DNA methylation (RdDM) functions in de novo meth-ylation 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 re-quired for CHH methylation on RdDM loci by transcribing long non-coding RNAs. Pol V influences the accumulation of 24-nucleotide small interfering RNAs (24-nt siRNAs) in a locus-specific manner. Bio-synthesis 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 meth-ylation levels in CG and CHG contexts predisposes 24-nt siRNA ac-cumulation 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.

Automated summary

This study reveals the essential role of rice Pol V in CHH methylation on RdDM loci, as well as its impact on the production and accumulation of 24-nt siRNAs. Pol V functions on loci with high CHH methylation levels and low CG and CHG methylation levels, while being not required on other loci.