Direct Read and Analysis of RNA Polymerase III derived RNAs
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PROVIDER: PRJEB81484 | ENA |
REPOSITORIES: ENA
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Project description:Many regulatory proteins and complexes have been identified which influence transcription by RNA polymerase (pol) II with a fine precision. In comparison, only a few regulatory proteins are known for pol III, which transcribes mostly house-keeping and non-coding RNAs. Yet, pol III transcription is precisely regulated under various stress conditions like starvation. We used proteomic approaches and pol III transcription complex components TFIIIC (Tfc6), pol III (Rpc128) and TFIIIB (Brf1) as baits to find identify the potential interactors through mass spectrometry-based proteomics. A large number of proteins were found in the interactome, which includes known chromatin modifiers, factors and regulators of transcription by pol I and pol II.
2019-01-02 | PXD003748 | Pride
Project description:There are about 600 loci in the mammalian genome that are annotated as RNA polymerase III genes. These comprise tRNA genes, the genes encoding 5S RNA, the smallest ribosomal RNA, and genes encoding catalytic or structural RNAs involved in processes as diverse as RNA processing or transcription elongation. Most RNA polymerase III genes have similar promoter structures, yet they are transcribed with different efficiencies. Here we have explored how RNA polymerase III occupancy of these genomic loci varies in a normal tissue, the liver, during the transition from a resting state to a proliferating state. We find that after partial hepatectomy, which causes synchronous entry of remaining liver cells into the cell division cycle, there is a tremendous increase in RNA polymerase III occupancy. This increase is, however, not uniform and concerns mostly loci that were lowly occupied by RNA polymerase III in resting liver. The changes in RNA polymerase III occupancy cannot be correlated with changes in RNA polymerase II occupancy around the RNA polymerase III loci nor at nearby RNA polymerase II promoters. RNA polymerase III loci with the largest fold change tend to be located in clusters, with the cluster displaying the largest changes located on chromosome 13. This suggests that increases in RNA polymerase III occupancy during the transition from resting to proliferating state affect mostly genes whose basal rate of transcription is relatively low and which are located in clusters.
2018-12-12 | GSE114650 | GEO
Project description:RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5´ peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential âhousekeepingâ roles. Many tRNA genes were found to generate long, 3´-extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of T-residues in the coding strand, and multiple, functional terminators can be located far downstream. The steady-state levels of 3´-extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery; especially the RNA-binding protein Nab2, cofactors for the nuclear exosome and the 5´-exonuclease Rat1. CRAC protocol performed on samples containing HTP-tagged proteins: Rpc160(Rpo31), Nab2, Rrp44(Dis3), Rrp6 and Mtr4. Rpc-160-bound RNAs were analyzed in wildtype and maf1 mutant cells and after a shift to medium containing glycerol.
2016-05-18 | E-GEOD-77863 | biostudies-arrayexpress
Project description:Adenovirus is a common human pathogen that relies on host cell processes for transcription and processing of viral RNA and protein production. Although adenoviral promoters, splice junctions, and cleavage and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and cleavage and polyadenylation sites across the adenovirus genome. In addition to confirming the known canonical viral early and late RNA cassettes, our analysis of splice junctions within long RNA reads revealed an additional 35 novel viral transcripts. These RNAs include fourteen new splice junctions which lead to expression of canonical open reading frames (ORF), six novel ORF-containing transcripts, and fifteen transcripts encoding for messages that potentially alter protein functions through truncations or fusion of canonical ORFs. In addition, we also detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking separate gene transcription units. Of these, an evolutionary conserved protein was detected containing the N-terminus of E4orf6 fused to the downstream DBP/E2A ORF. Loss of this novel protein, E4orf6/DBP, was associated with aberrant viral replication center morphology and poor viral spread. Our work highlights how long-read sequencing technologies can reveal further complexity within viral transcriptomes.
2022-08-30 | PXD034464 | Pride
Project description:RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5´ peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential “housekeeping” roles. Many tRNA genes were found to generate long, 3´-extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of T-residues in the coding strand, and multiple, functional terminators can be located far downstream. The steady-state levels of 3´-extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery; especially the RNA-binding protein Nab2, cofactors for the nuclear exosome and the 5´-exonuclease Rat1.
2016-05-18 | GSE77863 | GEO
Project description:RNA polymerase (Pol) III transcribes many noncoding RNAs (for example, transfer RNAs) important for translational capacity and other functions. We localized Pol III, alternative TFIIIB complexes (BRF1 or BRF2) and TFIIIC in HeLa cells to determine the Pol III transcriptome, define gene classes and reveal 'TFIIIC-only' sites. Pol III localization in other transformed and primary cell lines reveals previously uncharacterized and cell type–specific Pol III loci as well as one microRNA. Notably, only a fraction of the in silico–predicted Pol III loci are occupied. Many occupied Pol III genes reside within an annotated Pol II promoter. Outside of Pol II promoters, occupied Pol III genes overlap with enhancer-like chromatin and enhancer-binding proteins such as ETS1 and STAT1. Moreover, Pol III occupancy scales with the levels of nearby Pol II, active chromatin and CpG content. These results suggest that active chromatin gates Pol III accessibility to the genome. Use of ChIP-seq to identify genomic regions bound by RNA Polymerase III machinery in multiple cell types as well as RNA-seq in HeLa for gene expression analysis. See GSE20609 for whole human genome raw Pol III ChIP-array data. See link below for supplementary methods and analysis.
2010-04-14 | E-GEOD-20309 | biostudies-arrayexpress
Project description:RNA polymerase (Pol) III transcribes many noncoding RNAs (for example, transfer RNAs) important for translational capacity and other functions. We localized Pol III, alternative TFIIIB complexes (BRF1 or BRF2) and TFIIIC in HeLa cells to determine the Pol III transcriptome, define gene classes and reveal 'TFIIIC-only' sites. Pol III localization in other transformed and primary cell lines reveals previously uncharacterized and cell type–specific Pol III loci as well as one microRNA. Notably, only a fraction of the in silico–predicted Pol III loci are occupied. Many occupied Pol III genes reside within an annotated Pol II promoter. Outside of Pol II promoters, occupied Pol III genes overlap with enhancer-like chromatin and enhancer-binding proteins such as ETS1 and STAT1. Moreover, Pol III occupancy scales with the levels of nearby Pol II, active chromatin and CpG content. These results suggest that active chromatin gates Pol III accessibility to the genome.
2010-04-14 | GSE20309 | GEO
Project description:RNA polymerase (Pol) I, II, and III are most commonly described as having distinct roles in synthesizing ribosomal RNA (rRNA), messenger RNA (mRNA), and specific small noncoding (nc)RNAs, respectively. This delineation of transcriptional responsibilities is not definitive, however, as evidenced by instances of Pol II recruitment to genes conventionally transcribed by Pol III, including the co-transcription of RPPH1 - the catalytic RNA component of RNase P. A comprehensive understanding of the interplay between RNA polymerase complexes remains lacking, however, due to limited comparative analyses for all three enzymes. To address this gap, we applied a uniform framework for quantifying global Pol I, II, and III occu- pancies that integrates currently available human RNA polymerase ChIP-seq datasets. Occupancy maps are combined with a comprehensive multi-class promoter set that includes protein-coding genes, noncoding genes, and repetitive elements. While our genomic survey appropriately identifies recruitment of Pol I, II, and III to canonical target genes, we unexpectedly discover widespread recruitment of the Pol III machinery to promoters of specific protein-coding genes, supported by colocalization patterns observed for several Pol III-specific subunits. We show that Pol III-occupied Pol II promoters are enriched for small, nascent RNA reads terminating in a run of 4 Ts, a unique hallmark of Pol III transcription termination and evidence of active Pol III activity at these sites. Pol III disruption differentially modulates the expression of Pol III-occupied coding genes, which are functionally enriched for ribosomal proteins and genes broadly linked to unfavorable outcomes in cancer. Our map also identifies additional, currently unannotated genomic elements occupied by Pol III with clear signatures of nascent RNA species that are sensitive to disruption of La (SSB) - a Pol III-related RNA chaperone protein. These findings revise our current understanding of the interplay between Pols II and III and identify potentially novel small ncRNAs with broad implications for gene regulatory paradigms and RNA biology.
2024-06-26 | GSE268497 | GEO
Project description:POLR3G and POLR3GL-RNA polymerase III target genes
| PRJNA208073 | ENA
Project description:RNA polymerase III regulation during mammalian tissue regeneration
| PRJNA472061 | ENA