Project description:Functional analysis of the Arabidopsis thaliana Pol V largest subunit, NRPE1, carboxy-terminal domain by whole genome bisulfite sequencing
Project description:Assessment of the Pol IV largest subunit, NRPD1, DeCL domain deletion construct to rescue Pol IV-dependent siRNAs in the nrpd1-3 mutant (sRNA).
Project description:The plant-specific DNA-dependent RNA polymerase V (Pol V) evolved from Pol II to function in an RNA-directed DNA methylation pathway. Here, we have identified targets of Pol V in Arabidopsis thaliana on a genome-wide scale using ChIP-seq of NRPE1, the largest catalytic subunit of Pol V. We found that Pol V is enriched at promoters and evolutionarily recent transposons. This localization pattern is highly correlated with Pol V-dependent DNA methylation and small RNA accumulation. We also show that genome-wide association of Pol V with chromatin is dependent on all members of a putative chromatin-remodeling complex termed DDR. Our study presents the first genome-wide view of Pol V occupancy and sheds light on the mechanistic basis of Pol V localization. Furthermore, these findings suggest a role for Pol V and RNA-directed DNA methylation in genome surveillance and in responding to genome evolution. For wild type plants (ecotype Columbia) and nrpe1 mutants whole-genome small RNA (sRNA-seq) and bisulfite sequencing (BS-seq) was performed. In addition whole genome chromatin immunoprecipitation (ChIP-seq) was performed on wild type (ecotype Columbia) plants as a negative control with experimentals consiting of wild type plants carrying a C-terminally epitope tagged (2XFLAG) NRPE1, as well as the NRPE1-FLAG construct in drd1, dms3, and rdm1 mutant backgrounds.
Project description:RNA polymerases IV and V (Pol IV and Pol V) are plant-specific polymerases required for the generation of noncoding RNAs in RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Their subunit compositions largely resemble that of Pol II. However, the mechanism and accessory factors involved in their assembly remain largely unknown. In this study, we performed a forward genetic screen and identified novel mutant alleles of MINIYO (IYO), QUATRE-QUART 2 (QQT2) and NUCLEAR RNA POLYMERASE B/D/E 11 (NRPB/D/E11) that are defective in RdDM. We found that Pol IV-dependent small interfering RNAs (siRNAs) and Pol V-dependent transcripts were greatly reduced in the mutants. NRPE1, the largest subunit of Pol V, dissociated from other Pol V subunits in the iyo and qqt2 mutants, suggesting the involvement of IYO and QQT2 in Pol V assembly. Furthermore, we found that IYO and QQT2 were mutually dependent for their binding to the NRPE3 subassembly to facilitate the assembly of Pol V holoenzyme. Our findings reveal IYO and QQT2 as cofactors for Pol V assembly and provide mechanistic insights into how RNA polymerases are assembled in Arabidopsis.
Project description:We report genome-wide detection of long noncoding RNA (lncRNA) generate by Pol V in transcriptional gene silencing in Arabidopsis thaliana. We further show that most of these transcripts are bound by AGO4. RNA immunoprecipitation followed by high-throughput sequencing (RIP-seq) RNA immunoprecipitation with an anti-AGO4 antibody was performed in one biological replicate and included Col-0, nrpe1, and ago4-1; RNA immunoprecipitation with an anti-NRPE1 (largest subunit of Pol V) was performed in two replicates; replicate 1 included Col-0 and nrpe1, replicated two included Col-0, nrpe1, ago4-1, and idn2-1
Project description:Plants bearing a sulfurea epiallele (TAB2sulf) were crossed with nrpe1 (largest polV subunit) mutants in Solanum Lycopersicum. 7 plants of a F3 progeny expected to be 100% TAB2sulf and segregating for the nrpe1 mutation were sequenced. 4 plants bear the nrpe1 mutation and 3 plants have NRPE1 WT alleles. The aim of the experiment was to compare sRNA accumulation between nrpe1 and WT F3 plants with particular interest on SlTAB2 (Solyc02g005200) which has been previously associated with paramutation in the sulfurea background. Both WT and nrpe1 F3 plants had methylation levels consistent with the TAB2sulf epiallele. nrpe1 and sulfurea parental controls are added in single replicates and a WT cv M82 plant is also sequenced as control. More replicates of WT and M82 are sequenced in a related experiment.
Project description:Assessment of the Pol IV largest subunit, NRPD1, DeCL domain deletion construct to rescue Pol IV-dependent DNA methylation in the nrpd1-3 mutant (Bisulfite-Seq).
Project description:RNA Polymerase V transcription recruits siRNA-Argonaute protein complexes to chromatin, thereby specifying sites of RNA-directed DNA methylation (RdDM) and transcriptional gene silencing in plants. The Pol V largest subunit, NRPE1, has an extensive carboxyl-terminal domain (CTD) that is dispensable for catalytic activity in vitro, yet essential in vivo. A CTD subdomain, DeCL, named for its similarity to a chloroplast protein, DEFECTIVE CHLOROPLASTS AND LEAVES, is required for Pol V function at virtually all loci, similar to mutants defective for Pol V recruitment. Deletions removing three other CTD subdomains affect overlapping subsets of loci, similar to mutants lacking proteins that bind Pol V or its transcripts. A yeast two-hybrid screen for CTD-interactors identified the 3-prime -> 5-prime exoribonuclease, RRP6L1 as an interactor with the DeCL subdomain and the adjacent QS subdomain, named for its numerous glutamine-serine (QS) repeats. These RRP6L1-binding subdomains immediately follow the Argonaute-binding subdomain. Experimental evidence indicates that RRP6L1 trims the 3-prime ends of Pol V transcripts sliced by ARGONAUTE 4 (AGO4), suggesting a model whereby the adjacent CTD subdomains enable the spatial and temporal coordination of AGO4 and RRP6L1 RNA processing activities.
Project description:In plants, the plant-specific RNA polymerase V (Pol V) transcripts non-coding RNA and provides a docking platform for the association of accessory proteins in the RNA-directed DNA methylation (RdDM) pathway. Various components were uncovered in the process of DNA methylation, but it is still not clear how the transcription of Pol V is regulated. Here, we found the conserved elongation factor, SPT6L, bound to thousands of intergenic regions in an RNA polymerase II (Pol II) independent manner. The intergenic enrichment of SPT6L, interestingly, co-occupied with the largest subunit of Pol V (NRPE1) and the mutation of SPT6L led to the reduction of DNA methylation but not Pol V enrichment. Furthermore, the association of SPT6L at Pol V loci was dependent on the Pol V associated factor, SPT5L, rather than the presence of Pol V, and the interaction between SPT6L and NRPE1 was compromised in spt5l. Finally, Pol V RIP-seq revealed that SPT6L is required to maintain the amount and length of Pol V transcripts. Our findings revealed the critical role of a Pol II conserved elongation factor in Pol V mediated DNA methylation and transcription, and shed light on the mutual regulation between Pol V and Pol II in plants.