Roles of RNA polymerase IV in gene silencing

Abstract

Eukaryotes typically have three multi-subunit enzymes that decode the nuclear genome into RNA: DNA-dependent RNA polymerases I, II and III (Pol I, II and III). Remarkably, higher plants have five multi-subunit nuclear RNA polymerases: the ubiquitous Pol I, II and III, which are essential for viability; plus two non-essential polymerases, Pol IVa and Pol IVb, which specialize in small RNA-mediated gene silencing pathways. There are numerous examples of phenomena that require Pol IVa and/or Pol IVb, including RNA-directed DNA methylation of endogenous repetitive elements, silencing of transgenes, regulation of flowering-time genes, inducible regulation of adjacent gene pairs, and spreading of mobile silencing signals. Although biochemical details concerning Pol IV enzymatic activities are lacking, genetic evidence suggests several alternative models for how Pol IV might function.

Figure 1

Catalytic subunits of DNA-dependent RNA polymerases (a) The largest and second-largest subunits form the catalytic center. The image is a surface rendering generated using the crystal coordinates for a yeast Pol II elongation complex determined by Westover, Bushnell and Kornberg (PDB:1R9T). Only the two largest Pol II subunits are shown. The DNA template strand is shown in blue, the non-template strand in green and the nascent RNA in red. (b) Domain structures of RNAP largest subunits. E.coli (Ec RPOC) and yeast Pol II largest subunits (Sc RPB1) are compared to the Arabidopsis largest subunits for Pol I (At NRPA1), Pol II (At NRPB1), Pol III (At NRPC1), Pol IVa (At NRPD1a) and Pol IVb (At NRPD1b). Positions of conserved domains A–H are highlighted. Numbers below Pol IV domains are % identities to corresponding Arabidopsis Pol II subunit domains. CTDs of yeast and Arabidopsis Pol II largest subunits have 26 or 39 copies, respectively, of a seven amino acid (heptad) repeat. The domain with similarity to the Defective Chloroplasts and Leaves gene (DeCL domain), present in the CTDs of the Pol IVa and Pol IVb largest subunits, is shown in green. The CTD of NRPD1b also includes a region rich in WG/GW motifs, overlapping ten imperfect 16 amino acid repeats, and a domain composed of alternating glutamines and serines (QS-rich domain). (c) Domain structures of RNAP second-largest subunits. E.coli (Ec RPOB) and yeast Pol II subunits (Sc RPB2) are compared to the Arabidopsis second-largest subunits for Pol I (At NRPA2) , Pol II (At NRPB2), Pol III (At NRPC2) or Pol IV (At NRPD2). Positions of conserved domains A–I are highlighted. Numbers below Pol IV domains are % identities to the corresponding Arabidopsis Pol II subunit domains.

Figure 2

A variety of proteins participate in Pol IVa-dependent silencing pathways. The figure shows a subset of the proteins that are involved in RdDM, nat-siRNA, lsiRNA, short-range silencing and long-distance silencing pathways. Proteins involved in the various pathways are linked by color-coded lines. The diagram does not imply the order of events, but illustrates the diversity of functional collaborations that are possible. Not all mutants have been tested in every pathway, therefore other potential connections may exist. However, the figure reflects the models provided by the authors of the studies discussed in the text.

Figure 3

Possible modes of Pol IVa function. Pol IVa may transcribe a specialized DNA template, such as methylated DNA (a) or single-stranded RNA transcripts derived from methylated DNA loci (b). Alternatively, Pol IVa may transcribe dsRNA generated from bidirectional transcripts, including transcripts of natural antisense gene pairs, or dsRNAs resulting from the annealing of long-distance mobile RNAs with target mRNAs (c and d). The model shown in D may account for the involvement of multiple dicers and multiple RDR inputs in the nat-siRNA and long-distance silencing pathways.