Abstract
In plants, nuclear multisubunit RNA polymerases IV and V are RNA Polymerase II-related enzymes that synthesize non-coding RNAs for RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Here, we tested the importance of the C-terminal domain (CTD) of Pol IV’s largest subunit given that the Pol II CTD mediates multiple aspects of Pol II transcription. We show that the CTD is dispensable for Pol IV catalytic activity and Pol IV termination-dependent activation of RNA-DEPENDENT RNA POLYMERASE 2, which partners with Pol IV to generate dsRNA precursors of the 24 nt siRNAs that guide RdDM. However, 24 nt siRNA levels decrease ∼80% when the CTD is deleted. RNA-dependent cytosine methylation is also reduced, but only ∼20%, suggesting that siRNA levels typically exceed the levels needed for methylation of most loci. Pol IV-dependent loci affected by loss of the CTD are primarily located in chromosome arms, similar to loci dependent CLSY1/2 or SHH1, which are proteins implicated in Pol IV recruitment. However, deletion of the CTD does not phenocopy clsy or shh1 mutants, consistent with the CTD affecting post-recruitment aspects of Pol IV activity at target loci.
Highlights
Multisubunit RNA polymerases IV and V are plant-specific enzymes that play non-redundant roles in RNA-directed DNA methylation (RdDM), a process important for silencing DNA viruses, transposons, repetitive elements and a subset of genes
The NRPD1 C-terminal domain (CTD) is dispensable for Pol IV subunit assembly, nuclear localization, RNA polymerase activity and transcriptional coupling with RDR2
To test whether the CTD is needed for catalytic activity, Pol IV–RDR2 complexes were affinity purified, by virtue of the FLAG epitope tags engineered at the C-termini of NRPD1 or NRPD1 CTD, and assayed side-by-side for Pol IV transcription, Pol IV termination and coupling of Pol IV termination with RNA second strand synthesis by RDR2
Summary
Multisubunit RNA polymerases IV and V are plant-specific enzymes that play non-redundant roles in RNA-directed DNA methylation (RdDM), a process important for silencing DNA viruses, transposons, repetitive elements and a subset of genes (reviewed in: 1,2). Multiple exon–intron junction positions within the NRPD1 and NRPE1 genes are identical to those in NRPB1, which encodes the Pol II largest subunit, indicative of their shared ancestry [19]. NRPB1 encodes a C-terminal domain (CTD) composed of a repeating seven amino acid motif that is highly conserved throughout eukaryotes [20]. These heptad repeats are missing in Pols IV and V [19,21,22]. The Pol IV largest subunit has a relatively short CTD consisting almost entirely of a Defective Chloroplasts and Leaves (DeCL) subdomain named for its sequence similarity to a small protein family implicated in chloroplast pre-rRNA processing [23,24]. A similar, but non-identical DeCL subdomain is present in the Pol V largest subunit CTD as one of
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
