RNA polymerases IV and V
Eukaryotes have three multisubunit, nuclear DNA-dependent RNA polymerases: RNA polymerase I (Pol I) transcribes large ribosomal RNAs, Pol II transcribes mRNA precursors, and Pol III transcribes tRNA and 5S rRNA. Plants have two additional RNA polymerases known as Pol IV and Pol V, both of which evolved from Pol II and are specialized for RNA-directed DNA methylation (RdDM). Pol II, Pol IV and Pol V each have 12 subunits, many of which are shared by the 3 polymerases, but each also has specialized subunits. Subunits are named nuclear RNA polymerase B (NRPB) for Pol II subunits, NRPD for Pol IV subunits and NRPE for Pol V subunits.
The largest subunits in Pol IV and Pol V are NRPD1 and NRPE1, respectively, and they bind to a shared subunit NRPD2/NRPE2 to form the catalytic cores. NRPD1 and NRPE1 differ from NRPB1 (which is the largest subunit of Pol II) through numerous substitutions or deletions of conserved amino acids in the catalytic centre and in their carboxy-terminal domains (CTDs) , which probably contribute to their specialized functions in RdDM. Whereas the CTD of NRPB1 comprises multiple copies of a heptapeptide repeat, these repeats are absent in the CTDs of NRPD1 and NRPE1, which contain a motif found in a group of proteins called defective chloroplasts and leaves (DeCL). Authentic DeCL proteins are involved in processing rRNA, but the function of the DeCL motif in NRPD1 and NRPE1 remains unknown8 . The extended CTD of NRPE1 also contains Trp-Gly or Gly-Trp repeats, which form an Argonaute (AGO) hook region that can bind to AGO4, thus contributing to the specific role of Pol V in siRNA-directed de novo methylation.
In Pol II, the NRPB1 and NRPB2 subunits combine with NRPB5 and NRPB9A or NRPB9B to create the ‘jaw’ region that grips DNA during transcription. In addition to differences between NRPE1 and NRPB1, unique contributions of NRPE5 and NRPE9B to Pol V function imply that it may be adapted for transcribing templates with specific structural features or chromatin modifications. In vitro, Pol IV and Pol V can carry out RNA-primed transcription of DNA and transcribe from bipartite RNA–DNA templates; in addition, Pol IV can transcribe bipartite RNA– RNA templates. Until the in vivo templates of Pol IV and Pol V are known, deviations from their traditional activities, such as acting as an unconventional endonuclease or exonuclease, and the possibility of extrachromosomal templates should be considered. Similarly to Pol II, Pol IV and Pol V may require factors that assist entry into the nucleus from the cytoplasm, in which the subunits are synthesized and assembled. The identification of homologues of yeast IWR1 (interacts with RNA polymerase II, which facilitates nuclear import of Pol II) in screens for RdDM-defective mutants indicates that Pol IV and Pol V might be imported into the nucleus in a similar way to that of Pol II.
A transcription fork model for RNA-directed DNA methylation (RdDM) is shown
In RNA polymerase IV (Pol IV)-dependent small interfering RNA (siRNA) biogenesis (left panel), Pol IV transcribes a single-stranded RNA (ssRNA) that is copied into a double-stranded RNA (dsRNA) by RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) with the assistance of the chromatin remodeller CLASSY 1 (CLSY1). The dsRNA is processed by DICER-LIKE 3 (DCL3) into 24-nucleotide siRNAs that are methylated at their 3ʹ ends by HUA ENHANCER 1 (HEN1) and incorporated into ARGONAUTE 4 (AGO4). SAWADEE HOMEODOMAIN HOMOLOGUE 1 (SHH1), which binds to histone H3 methylated at lysine 9 (H3K9me), interacts with Pol IV and recruits it to some target loci.
In Pol V-mediated de novo methylation (middle panel), Pol V transcribes a scaffold RNA that base-pairs with AGO4-bound siRNAs. AGO4 is recruited through interactions with the AGO hook regions in the carboxy-terminal domain of the largest subunit of Pol V and with KOW DOMAIN-CONTAINING TRANSCRIPTION FACTOR 1 (KTF1). RNA-DIRECTED DNA METHYLATION 1 (RDM1) links AGO4 and DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which catalyses de novo methylation of DNA. Pol V transcription may be enabled by the duplex unwinding activity of the chromatin remodeller DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1), whereas the single-stranded DNA-binding activity of RDM1 and the putative cohesin-like roles of DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) and MICRORCHIDIA 6 (MORC6) may help to generate and stabilize the unwound state. Pol V recruitment is potentially aided by SUVH2 or SUVH9, both of which bind to methylated DNA.
Nucleosome positioning (right panel) is adjusted by the SWI/SNF complex, which interacts with the IDN2 (INVOLVED IN DE NOVO 2)–IDP (IDN2 PARALOGUE) complex that binds to Pol V scaffold RNAs. Deposition of repressive histone modifications — such as H3K9me by SUVH4, SUVH5 and SUVH6 — is facilitated following removal of active marks by HISTONE DEACETYLASE 6 (HDA6), JUMONJI 14 (JMJ14) and UBIQUITIN-SPECIFIC PROTEASE 26 (UBP26). Higher-order chromatin conformations that reinforce the silent state are established through the ATPase activities of MORC1 and MORC6 (not shown). Adapted with permission from REF148., Cold Spring Harbor Laboratory Press.
example,
Non-canonical Pol II–RDR6-dependent RdDM pathway
This pathway provides a means to establish RNA-directed DNA methylation (RdDM) and eventually ensure stable transcriptional gene silencing (TGS) of a newly acquired transposon that is originally a target of post-transcriptional gene silencing (PTGS).
In PTGS (left panel), a newly inserted transposon is initially active and transcribed by RNA polymerase II (Pol II). Some of the transcripts are copied by RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) to produce double-stranded RNAs (dsRNAs), which are processed by DICER-LIKE 2 (DCL2) and DCL4 into 21–22-nucleotide (nt) small interfering RNAs (siRNAs). These siRNAs are loaded onto ARGONAUTE 1 (AGO1) and guide cleavage of transposon transcripts in a typical PTGS pathway.
In a deviation from the canonical RdDM pathway (middle panel), some of the 21–22-nt siRNAs can also trigger low levels of DNA methylation in a manner that is dependent on DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), Pol V and AGO2, which interacts with NEEDED FOR RDR2-INDEPENDENT DNA METHYLATION (NERD) through its AGO hook motif. The sparsely methylated DNA recruits Pol IV, which initiates the canonical RdDM pathway by transcribing a single-stranded RNA (ssRNA). The ssRNA is copied by RDR2 into a dsRNA that is processed by DCL3 into 24-nt siRNAs. Following incorporation into AGO4 (right panel), the 24-nt siRNAs base-pair with Pol V scaffold transcripts, which results in DRM2 recruitment and dense methylation. siRNAs are continuously produced from the methylated template by Pol IV pathway components, which reinforces TGS that can be maintained in an siRNA-independent manner by METHYLTRANSFERASE 1 (MET1), CHROMOMETHYLASE 3 (CMT3) and DECREASED DNA METHYLATION 1 (DDM1)(not shown).
lease of RdDM during stress
a | A schematic locus is shown, in which a silenced transposon leads to the silencing of a nearby protein-coding gene (Gene 1). By contrast, the activity of other protein-coding genes (Gene 2 and Gene 3) are not under control of the transposon.
b | Transposons are activated during biotic and abiotic stress responses through a combination of loss of RNA-directed DNA methylation (RdDM) and transcriptional responses to stress . The nearby Gene 1 is also activated owing to loss of methylation in promoter regions . Reactivated transposons can integrate into new genomic loci, although the RdDM machinery inhibits the reinsertion of some elements through an unknown mechanism.
c | New transposon insertions can establish stress-responsive transcription at additional protein-coding genes (Gene 2) or might permanently disrupt gene function (Gene 3).
logical processes involving siRNAs and RdDM components
a | RNA-directed DNA methylation (RdDM) is implicated in defence against some viral and bacterial pathogens, and affects the development of pathogenic tumours. Biotic and abiotic stress responses include RdDM-mediated changes in DNA methylation.
b | High copy-number transposable elements (shown in blue) are transcriptionally silenced by RdDM, which promotes genome stability.
c | Small interfering RNAs (siRNAs) and the RdDM machinery are also involved in specification of female germ cells and might be involved in establishment or maintenance of parent-of-origin genomic imprints.
d | By initiating methylation that can be passed to progeny and stably maintained, RdDM might establish epialleles. Conversely, production of siRNAs can remethylate DNA after loss of epigenetic marks.
e | siRNAs can also mediate interactions between maternal and paternal genomes upon hybridization, particularly at sites of paramutation. RdDM can mediate both allelic and non-allelic communication within a cell.
f | Alternatively, RdDM can function in a non-cell autonomous way to communicate information between cells. In reproductive tissues, siRNA movement might occur between the pollen vegetative nucleus and the sperm cells (blue), between somatic tissues and the megaspore mother cell (pink), or between the endosperm and the embryo after fertilization (green).
g | Intercellular transport of siRNAs in vegetative tissues is likely to occur through cytoplasmic tunnels that connect neighbouring cells.
h | Intercellular transport of siRNAs can eventually occur over long distances and bidirectionally through the vascular system to induce methylation at remote sites.
