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

Abstract

Multisubunit RNA polymerase (Pol) complexes are the core machinery for gene expression in eukaryotes. The enzymes Pol I, Pol II and Pol III transcribe distinct subsets of nuclear genes. This family of nuclear RNA polymerases expanded in terrestrial plants by the duplication of Pol II subunit genes. Two Pol II-related enzymes, Pol IV and Pol V, are highly specialized in the production of regulatory, non-coding RNAs. Pol IV and Pol V are the central players of RNA-directed DNA methylation (RdDM), an RNA interference pathway that represses transposable elements (TEs) and selected genes. Genetic and biochemical analyses of Pol IV/V subunits are now revealing how these enzymes evolved from ancestral Pol II to sustain non-coding RNA biogenesis in silent chromatin. Intriguingly, Pol IV-RdDM regulates genes that influence flowering time, reproductive development, stress responses and plant-pathogen interactions. Pol IV target genes vary among closely related taxa, indicating that these regulatory circuits are often species-specific. Data from crops like maize, rice, tomato and Brassicarapa suggest that dynamic repositioning of TEs, accompanied by Pol IV targeting to TE-proximal genes, leads to the reprogramming of plant gene expression over short evolutionary timescales.

Keywords: RNA polymerase IV (Pol IV); RNA-directed DNA Methylation; non-coding RNA; plant gene regulation; transposable elements.

Figure 1.

Schematic overview of the RNA-directed DNA methylation (RdDM) pathway. (a) Pol IV transcripts are processed by RDR2 into double-stranded RNA (dsRNA), which is diced into a 24 nt small interfering RNA (siRNA) duplex by DCL3. HEN1 performs 2'O-methylation of each siRNA strand. AGO4-siRNA complexes bind to complementary sequences in nascent Pol V transcripts, and this AGO4-siRNA-Pol V complex is stabilized by the interaction of the NRPE1 (Pol V) CTD with AGO4, and of Pol V with SPT5L. Finally, this leads to the recruitment of DRM2, which catalyzes de novo cytosine methylation. (b) The Pol IV-RdDM pathway is initiated by recruitment of Pol IV to silent chromatin; this typically occurs in distal chromosomal regions by the dimethylated Histone 3 Lysine 9 (H3K9me2) reader, SHH1, which interacts with Pol IV through chromatin remodelers CLSY1 or CLSY2. In pericentromeric regions, CLSY3 and CLSY4 are required for Pol IV recruitment, which may interact with these DNA regions using a DNA methylation reader, so far unknown in a direct or indirect fashion. (c) Pol V is recruited to chromosomal targets by a dedicated machinery, mostly different from the factors required for Pol IV transcription. SUVH2 and SUVH9 are SET and RING-associated (SRA) domain proteins thought to recruit Pol V to regions of methylated DNA. The DDR complex (DRD1, DMS3 and RDM1; not detailed here) serves as a bridge complex that mediates Pol V transcription at many, if not all RdDM targets. Pol V interactions with the target DNA and chromatin are further consolidated by MORC6

Figure 2.

Pol IV and Pol V evolved from Pol II but have specific subunits and domains. (a) Pol II, Pol IV and Pol V are composed of 12 subunits (respectively called NRPB, NRPD and NRPE from 1 to 12). Certain subunits are common to all three complexes (yellow); others are unique to Pol II (green), Pol IV (purple) or Pol V (blue); and a few assemble with Pol IV and Pol V but not with Pol II (pink). The 4^th and 7^th subunits of nuclear RNA polymerases form a stalk domain. (b) NRPB1, NRPD1 and NRPE1, the largest subunits of Pol II, Pol IV and Pol V, contain several conserved domains (A to H). NRPB1 contains specific domains (green): the bridge helix, trigger loop (lost in NRPD1 and NRPE1) and heptad repeats in its carboxy-terminal domain (CTD). The DEFECTIVE CHLOROPLASTS AND LEAVES (DeCL) domain is common to both Pol IV and Pol V (red). NRPD1 contains a specific CKYC-YP motif between the A and the B domain (purple). The NRPE1 CTD contain WG motifs and a SQ-rich domain (blue)

Figure 3.

The genome surveillance function of RNA polymerase IV (Pol IV) has been coopted for plant gene regulation. (a) Evolutionary tree representing the plant species reported to have de novo DNA methylation by Pol IV and RNA-directed DNA Methylation (RdDM). Dashed lines indicate the predicted timescale of plant diversification in million years (My) [208]. Highlighted in green are the species in which genes encoding RdDM players are present. Red indicates that no evidence for genes encoding RdDM factors has been reported. (b) Tandem repeats similar to transposable elements (TEs) in the FWA gene promoter allow Pol IV and RdDM to repress FWA expression for flowering time regulation in the Arabidopsis genus. (c) Insertion of Miniature Inverted-repeat Transposable Elements (MITEs) near gene loci can regulate gene expression in Oryza sativa (rice). Pol IV represses a miRNA precursor gene, OsMIR156j, in wild-type rice. Transcriptional silencing of OsMIR156j is disrupted in Os nrpd1a Os nrpd1b mutant plants, causing miR156 to overaccumulate and target the mRNA of Os IPA1, which ultimately leads to increased tillering. (d) Plant siRNAs can also target and transcriptionally repress genes in trans, for example to regulate innate immunity in rice. Expression of the Os STI gene leads to Xanthomonas oryzae pv. oryzae (Xoo) resistance. In Xoo susceptible plants harboring the WRKY45-1 allele, an intronic MITE triggers production of siRNAs via Pol IV-RdDM that will guide DNA methylation to a homologous MITE sequence in an intron of STI gene and silence it. In Xoo resistant plant harboring the WRKY45-2 allele, the intronic MITE and resultant silencing of STI is missing. (e) In maize, paramutation depends on the Pol IV and RDR2 enzyme machinery for production of 24 nt siRNAs. The parental “paramutagenic” B’ allele is linked to a silent booster 1 (b1) locus, whereas b1 is still expressed in the case of a B-I “paramutable” allele. When B-I/B-I (purple) and B’/B’ (green) individuals are crossed to form the B-I/B’ genotype in F1 plants, siRNAs from B’ are thought to silence the B-I allele in trans, thereby changing B-I into silent B’* and shutting down anthocyanin production. The DNA methylation induced by B’ is heritable to all F2 progeny (B’*/B’*, B’*/B’, B’/B’* or B’/B’ genotypes), and newly formed B’* alleles are also paramutagenic in future crosses

Figure 4.

Factors that regulate, initiate and counterbalance Pol IV function in RdDM. (a) Components of the Pol IV-RdDM machinery could be regulated by transcriptional control of Pol IV subunit/partner genes, post-transcriptional silencing of the corresponding mRNAs, post-translational modification of the individual proteins, or targeted degradation of the Pol IV complex itself. Pol IV assembly and turnover is likely governed by the specific subunits and functional domains that mediate Pol IV’s interactions with SHH1, CLSYs, RDR2 and yet unknown, specialized regulatory proteins. (b) Initial recruitment of Pol IV to specific sites in the genome may require factors other than SHH1 and CLSY proteins, which still remain to be discovered. Another important process that controls the intensity of Pol IV-RdDM is the balance between CG/CHG methylation maintenance (involving MET1, CMT3, and HDA6 proteins), and active 5-methylcytosine removal by plant glycosylase lyases (ROS1 and DME). (c) In addition to the canonical RdDM pathway involving Pol IV-RDR2-DCL3 and 24 nt siRNAs loaded onto AGO4 (bold arrows), other pathways can trigger de novo DNA methylation in plants (thin arrows). The alternatives include Pol II-RDR6 production of dsRNAs that are diced into 21–22 nt siRNAs, or Pol IV-RDR2 production of dsRNAs that are diced by the alternate enzymes DCL2 and DCL4, into 22 and 21 nt siRNAs, which tend to associate with different effectors, such as AGO1 and AGO2, to guide DNA methylation