The RPB2 flap loop of human RNA polymerase II is dispensable for transcription initiation and elongation.
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE29734 | GEO | 2011/06/24
SECONDARY ACCESSION(S): PRJNA141155
REPOSITORIES: GEO
Similar Datasets
Project description:Deletion of the flap loop in the Rp2 subunit of human RNAPII did not alter the ability of human RNAPII to initiate transcription or elongate the initiated transcripts in vivo and in vitro, and was also dispensable for elongation factor-mediated enhancement and inhibition of transcript elongation in vitro. ChIP:chip of wild-type and flap deletion human RNAPII revealed that the mutant RNAPII was not defective for promoter binding, initiation and elongation in vivo. Eight ChIP datasets were generated as log2(IP/input) fluorescence intensities using anti-FLAG IP of wild-type RPB2-FLAG cells in biological triplicate, anti-FLAG IP of delta-FL RPB2 FLAG cells in biological triplicate, and anti-RPB1 CTD, 8WG16 of both wild-type and delta-FL cells in biological duplicate. The two 8WG16- IP datasets were combined to measure the distribution of RPB1 signal.
2011-06-24 | E-GEOD-29734 | biostudies-arrayexpress
Project description:Single stranded DNA binding proteins play many roles in nucleic acid metabolism, but their importance during transcription remains unclear. Quantitative proteomic analysis of RNA polymerase II (RNApII) pre-initiation complexes (PICs) identified Sub1 and the Replication Protein A complex (RPA), both of which bind single-stranded DNA (ssDNA). Sub1, homolog of mammalian coactivator PC4, exhibits strong genetic interactions with factors necessary for promoter melting. Sub1 localizes near the transcription bubble in vitro and binds to promoters in vivo dependent upon PIC assembly. In contrast, RPA localizes to transcribed regions of active genes, strongly correlated with transcribing RNApII but independently of replication. RFA1 interacts genetically with transcription elongation factor genes. Interestingly, RPA levels increase at active promoters in cells carrying a Sub1 deletion or ssDNA binding mutant, suggesting competition for a common binding site. We propose that Sub1 and RPA interact with the non-template strand of RNApII complexes during initiation and elongation, respectively. Chip-chip from wt and sub1D cells with Rfa1 Chromatin immunoprecipitation (ChIP) of Rfa1 in wt and sub1D yeast demonstrated that Rfa1 localization correlates with RNA Polymerase II and is increased at some transcription start sites when Sub1 has been deleted. Comparison of Rfa1 localization in wt vs sub1D yeast
2011-09-26 | E-GEOD-32416 | biostudies-arrayexpress
Project description:The production of functional mRNA involves multiple steps including transcription initiation, elongation, and termination. spt5 encodes a conserved essential transcription elongation factor that controls RNAPII processivity in vitro and co-localizes with RNAPII in vivo. We used microarrays to detail the global expression of gene expression at 24 hours post fertilization in wild-type and Spt5 mutant (fog-sk8) to identify Spt5 transcriptionally dependent genes and affected pathways. Here we report that a mutation in zebrafish spt5, sk8, deregulates <5% of ~10,000 genes examined. These findings reveal a small but diverse set of developmentally regulated genes, whose expression is critically dependent on Spt5-regulated RNAPII stalling in vertebrate development. Experiment Overall Design: RNA from zebrafish embryos were harvested at 24 hours post fertilization for hybridization to Affymetrix gene arrays.
2008-10-25 | E-GEOD-12826 | biostudies-arrayexpress
Project description:Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative splicing. Pausing sites and changes in the rate of transcription by RNAPII may therefore have a fundamental impact in the regulation of alternative splicing. Here, we show that the elongation and splicing-related factor TCERG1 regulates alternative splicing of the apoptosis gene Bcl-x in a promoter-dependent manner. TCERG1 promotes the splicing of the short isoform of Bcl-x (Bcl-xs) through the SB1 regulatory element located in the first half of exon 2. Consistent with these results, we show evidence for in vitro and in vivo interaction of TCERG1 with the Bcl-x pre-mRNA. Transcription profile analysis reveals that the RNA sequences required for the effect of TCERG1 on Bcl-x alternative splicing coincide with a putative polymerase pause site. Furthermore, TCERG1 modifies the impact of a slow polymerase on Bcl-x alternative splicing. In support of a role for an elongation mechanism in the transcriptional control of Bcl-x alternative splicing, we found that TCERG1 modifies the amount of pre-mRNAs generated at distal regions of the endogenous Bcl-x. Most importantly, TCERG1 affects the rate of RNAPII transcription of endogenous human Bcl-x. We propose that TCERG1 modulates the elongation rate of RNAPII to relieve pausing, thereby activating the pro-apoptotic Bcl-xS 5’ splice site. ChIP-Seq
2012-01-12 | E-GEOD-33716 | biostudies-arrayexpress
Project description:Elongin is an RNA polymerase II (RNAPII)-associated factor that has been shown to stimulate transcriptional elongation in vitro. The Elongin complex is thought to be required for transcriptional induction in response to cellular stimuli and to ubiquitinate RNAPII in response to DNA damage. Yet the impact of the Elongin complex on transcription in vivo has not been well studied. Here, we performed comprehensive studies of the role of Elongin A, the largest subunit of the Elongin complex, on RNAPII transcription genome-wide.
In an effort to explore regulatory roles for Elongin A, we performed IP-MS. We constructed a DLD1 cell line expressing Flag-tagged Elongin A at endogenous levels and performed anti-Flag immuno-purification of solubilized chromatin followed by analysis of binding partners by mass spectrometry in triplicates. We identified a large group of Elongin A-associated proteins. We confirmed a subset of possible interacting proteins by Co-IP and western blotting. Consistent with previous studies, we identified RNA Pol II subunits and proteins related to transcription elongation and RNA processing. Among the top hits, we identified nearly all subunits of the PAF1 complex, except for RTF1, which does not stably associate with mammalian PAF1. Concordantly, in our previous PAF1 proteomic study, Elongin A was also among the top hits. We also identified several subunits of the Integrator complex, a multi-subunit complex that has been shown to participate in enhancer RNA (eRNA) processing, a finding consistent with our conclusion that Elongin A localizes to potential enhancers. Collectively, our results suggest that Elongin A stably interacts with RNAPII and the transcription machinery on chromatin.
Taken together, our studies suggest that Elongin A associates with the transcription machinery at actively transcribed genomic regions and may be involved in the release of paused RNAPII. However, Elongin A does not appear to be critical for maintaining transcription elongation rates in vivo.
2020-08-07 | MSV000085926 | MassIVE
Project description:Dynamic control of gene expression is crucial for most aspects of cell physiology and at its core is RNA polymerase II (RNAPII). Using 53 RNAPII point mutants, we generated a point mutant epistatic miniarray profile (pE-MAP) comprising ~60,000 quantitative genetic interactions in Saccharomyces cerevisiae. This enabled functional assignment of RNAPII sub-domains, including connections to protein complexes. Using splicing microarrays and point mutants altering elongation rates in vitro, we found an inverse relationship between RNAPII speed and in vivo splicing efficiency. Furthermore, the pE-MAP classified groups of fast and slow mutants that favor upstream and downstream start site selection, respectively. Finally, the pE-MAP identified Sub1 as a positive transcription factor regulating start site selection and splicing. These data reveal striking coordination of polymerization rate with transcription initiation and splicing, suggesting transcription rate is tuned to coordinate multiple gene expression steps. The pE-MAP approach provides a powerful strategy to understand other multi-functional machines at amino acid resolution. Two channel microarrays were used. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used in one of the channels for each hybridization and used in the statistical analysis to obtain an average expression-profile for each deletion mutant relative to the wt. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Using the Erlenmeyer growth protocol up to five deletion strains were grown on a single day. In the tecan platereader, up to eleven deletion strains could be grown on a single day. Wt cultures were grown parallel to the deletion mutants to assess day-to-day variance. Strains with point mutations in either rpb1, rpb2 or rpb7 subunits of RNA polymerase II (RNAPII) examined under normal growth conditions. The mutated RNAPII subunit is present on URA3 or LEU2 marked CEN plasmid, and the corresponding genomic RNAPII subunit deleted. Strains labeled as wildtype also have the relevant genomic RNAPII subunit deleted, but complemented with wildtype RNAPII subunit on CEN plasmid.
2013-08-06 | E-GEOD-47429 | biostudies-arrayexpress
Project description:Single stranded DNA binding proteins play many roles in nucleic acid metabolism, but their importance during transcription remains unclear. Quantitative proteomic analysis of RNA polymerase II (RNApII) pre-initiation complexes (PICs) identified Sub1 and the Replication Protein A complex (RPA), both of which bind single-stranded DNA (ssDNA). Sub1, homolog of mammalian coactivator PC4, exhibits strong genetic interactions with factors necessary for promoter melting. Sub1 localizes near the transcription bubble in vitro and binds to promoters in vivo dependent upon PIC assembly. In contrast, RPA localizes to transcribed regions of active genes, strongly correlated with transcribing RNApII but independently of replication. RFA1 interacts genetically with transcription elongation factor genes. Interestingly, RPA levels increase at active promoters in cells carrying a Sub1 deletion or ssDNA binding mutant, suggesting competition for a common binding site. We propose that Sub1 and RPA interact with the non-template strand of RNApII complexes during initiation and elongation, respectively.
2011-09-27 | GSE32416 | GEO
Project description:Here we characterized a genetic regulatory system named as the hpDNA-assisted structure-guided nuclease mediating interference (HpSGNi) system, which was composed of flap endonuclease 1 (FEN1) and mis-hairpin DNA probe (mis-hpDNA). To investigate gene regulation effective of the HpSGNi system, we co-transformed HEK293A (human embryonic kidney epithelial cells) with plasmids encoding FEN1 with NES and two groups of mis-hpDNAs targeting the EGFP gene and negative control, respectively. RNA-seq was used to examine the regulation effective. We proved the HpSGNi system can knock down specific gene in human cells. At the same time, it indicated that a degree of off-targets occur in HpSGNi-treated cells.
2024-04-11 | GSE171834 | GEO
Project description:Transcriptional elongation factor Elongin A exhibits an activity to repress the temporary pause of RNA polymerase II (RNAPII) on the DNA template, resulting in increase of mRNA synthesis in vitro. However, actual elongation ability of Elongin A in mammalian cells has not been determined yet. To elucidate the practical transcriptional role of mammalian Elongin A in vivo, we carried out whole genome expression analysis using Elongin A knocked-down HeLa cells. Gene expression in Elongin A knocked-down HeLa cells was measured at 2 hours after doxorubicin treatment (1microM).
2013-09-03 | E-GEOD-30432 | biostudies-arrayexpress
Project description:As a central component during Okazaki fragment maturation, flap endonuclease 1 (FEN1) removes the 5â flap and maintains genomic stability. Here, FEN1 was cloned as a suppressor of transcriptional gene silencing (TGS) from a forward genetic screen. FEN1 is abundant in the root and shoot apical meristems and FEN1-GFP shows a nucleolus-localized signal in tobacco cells. Arabidopsis fen1-1 mutant is hypersensitive to MMS and shows reduced telomere length. Interestingly, genome-wide ChIP-seq and RNA-seq results demonstrate that FEN1 mutation leads to a decrease in the H3K27me3 level and an increase in the expression of a subset of genes marked with H3K27me3. Overall, these results uncover a role for FEN1 in mediating TGS besides maintaining genome stability in Arabidopsis. To characterized the role of FEN1 in epigenetic silencing, we examine histone modification and RNA expression changes by whole-genome RNA sequencing; H3K27me3-, H3K4me3-, H3K9me2-, H3-ChIP-seq in A. thaliana transgenic wild type (TWT) and fen1 mutant
2016-05-28 | E-GEOD-79738 | biostudies-arrayexpress