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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.
Pubmed ID: 31329950
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Software environment and programming language for statistical computing and graphics. R is integrated suite of software facilities for data manipulation, calculation and graphical display. Can be extended via packages. Some packages are supplied with the R distribution and more are available through CRAN family.It compiles and runs on wide variety of UNIX platforms, Windows and MacOS.
View all literature mentionsAn R package for DNA methylation analysis and annotation from high-throughput bisulfite sequencing.
View all literature mentionsSoftware tool to map bisulfite converted sequence reads and determine cytosine methylation states. Flexible aligner and methylation caller for Bisulfite-Seq applications. Used to map bisulfite treated sequencing reads to genome of interest and perform methylation calls in single step.
View all literature mentionsA software tool developed to process and analyze small RNA-seq data with respect to a reference genome, and output a comprehensive and informative annotation of all discovered small RNA genes. ShortStack discovers small RNA ''clusters'' de novo, based on user-set thresholds, and annotates clusters with respect to small RNA size, orientation, and repetitiveness. ShortStack also discovers and annotates MIRNA genes, and other Hairpin-associated small RNA genes. In addition, ShortStack includes a robust method to detect genes producing small RNAs in a phased manner. It outputs a descriptive table of all results, useful genome browser tracks, a table describing the results of the hairpin / MIRNA analysis for each cluster, and detailed text-based alignments of all MIRNAs and hairpin-associated clusters. It can also be run in ''count'' mode, to quantify a set of input loci with genomic coordinates determined a priori by the user. ShortStack is a perl program. Besides perl, ShortStack also requires samtools and the RNALfold and RNAeval programs from the Vienna RNA Package to execute. When used to control the alignment of small RNA data to a reference genome, ShortStack also requires bowtie and bowtie-build. Finally, for optimal results, ShortStack uses a file of inverted repeats produced by the EMBOSS application einverted.
View all literature mentionsSoftware tool that removes adapter sequences from DNA sequencing reads.
View all literature mentionsConversion software that both demultiplexes data and converts BCL files generated by Illumina sequencing systems to standard FASTQ file formats for downstream analysis.
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