The NRPD1 N-terminus contains a Pol IV-specific motif that is critical for genome surveillance in Arabidopsis

Abstract

RNA-guided surveillance systems constrain the activity of transposable elements (TEs) in host genomes. In plants, RNA polymerase IV (Pol IV) transcribes TEs into primary transcripts from which RDR2 synthesizes double-stranded RNA precursors for small interfering RNAs (siRNAs) that guide TE methylation and silencing. How the core subunits of Pol IV, homologs of RNA polymerase II subunits, diverged to support siRNA biogenesis in a TE-rich, repressive chromatin context is not well understood. Here we studied the N-terminus of Pol IV’s largest subunit, NRPD1. Arabidopsis lines harboring missense mutations in this N-terminus produce wild-type (WT) levels of NRPD1, which co-purifies with other Pol IV subunits and RDR2. Our in vitro transcription and genomic analyses reveal that the NRPD1 N-terminus is critical for robust Pol IV-dependent transcription, siRNA production and DNA methylation. However, residual RNA-directed DNA methylation observed in one mutant genotype indicates that Pol IV can operate uncoupled from the high siRNA levels typically observed in WT plants. This mutation disrupts a motif uniquely conserved in Pol IV, crippling the enzyme's ability to inhibit retrotransposon mobilization. We propose that the NRPD1 N-terminus motif evolved to regulate Pol IV function in genome surveillance.

Epigenetic silencing of clustered tRNA genes in Arabidopsis

“…In silico analyses of epigenomic landscapes show that silencing of clustered tDNAs is correlated with particular epigenetic states involving DNA methylation and repressive histone marks. As compared to dispersed tDNAs scattered in euchromatic environment, clustered tDNAs display high CG methylation levels suggesting that DNA methylation triggers their transcriptional repression as described for most repeats and TEs in Arabidopsis ( 79 , 80 ). Indeed, epigenetic silencing at tDNA clusters is under the complex interplay of DNA methyltransferases MET1, CMT2 and CMT3, as well as chromatin remodeling factor DDM1 (Figure 7 ).…”

Assembly of a double-stranded RNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 docks with NUCLEAR RNA POLYMERASE IV at the clamp domain

“…These results suggest that RDR2 can bind independently to either side of the putative zinc finger in a sequence-specific manner, consistent with the conservation of sequences adjacent to the CxxC motifs (see Figure 6C). Moreover, a tyrosine substitution at the first cysteine of the CxxC motif at the beginning of RDR2 interaction site 2 (C118Y, defining mutant allele nrpd1-50) did not disrupt RDR2 interaction, despite severly impairing Pol IV activity in vivo and in vitro (39). Thus the putative zinc finger may play a role in Pol IV transcription independent of RDR2 interaction.…”

Non-coding RNA polymerases that silence transposable elements and reprogram gene expression in plants

“…We recently discovered that Pol IV harbors a novel amino acid motif in its NRPD1 N-terminus, which is absent in Pol II but highly conserved in Pol IV [ 102 ]. This motif is composed of a C[KR]YC box followed by a 5–10 amino acid spacer, then by a YPx[MV][KR]F[KR] box ( Figure 2b ).…”

“…This motif is composed of a C[KR]YC box followed by a 5–10 amino acid spacer, then by a YPx[MV][KR]F[KR] box ( Figure 2b ). A point mutation in the motif caused a loss in 24 nt siRNA biogenesis, disrupted de novo DNA methylation and reactivated TE loci in A. thaliana [ 102 ]. Beyond its critical role in genome surveillance and DNA methylation patterning, the precise function of the motif is not fully understood.…”

“…Residual 24 nt siRNAs accumulate at TE extremities and other hotspots in the epigenomic landscape of the C[KR]YC-box mutant, suggesting that these could be sites of RdDM initiation. One attractive model is that the Pol IV-specific motif in NRPD1’s N-terminus governs the mechanism of silent chromatin amplification as RdDM spreads across a locus in WT plants [ 102 ].…”