scholarly journals The conserved ribonuclease aCPSF1 triggers genome-wide transcription termination of Archaea via a 3′-end cleavage mode

2020 ◽  
Vol 48 (17) ◽  
pp. 9589-9605 ◽  
Author(s):  
Lei Yue ◽  
Jie Li ◽  
Bing Zhang ◽  
Lei Qi ◽  
Zhihua Li ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Termination ◽  
Methanococcus Maripaludis ◽  
Genome Wide ◽  
Transcription Termination Factor ◽  
A Genome ◽  
Archaeal Transcription ◽  
Archaeal Ancestor

Abstract Transcription termination defines accurate transcript 3′-ends and ensures programmed transcriptomes, making it critical to life. However, transcription termination mechanisms remain largely unknown in Archaea. Here, we reported the physiological significance of the newly identified general transcription termination factor of Archaea, the ribonuclease aCPSF1, and elucidated its 3′-end cleavage triggered termination mechanism. The depletion of Mmp-aCPSF1 in Methanococcus maripaludis caused a genome-wide transcription termination defect and disordered transcriptome. Transcript-3′end-sequencing revealed that transcriptions primarily terminate downstream of a uridine-rich motif where Mmp-aCPSF1 performed an endoribonucleolytic cleavage, and the endoribonuclease activity was determined to be essential to the in vivo transcription termination. Co-immunoprecipitation and chromatin-immunoprecipitation detected interactions of Mmp-aCPSF1 with RNA polymerase and chromosome. Phylogenetic analysis revealed that the aCPSF1 orthologs are ubiquitously distributed among the archaeal phyla, and two aCPSF1 orthologs from Lokiarchaeota and Thaumarchaeota could replace Mmp-aCPSF1 to terminate transcription of M. maripaludis. Therefore, the aCPSF1 dependent termination mechanism could be widely employed in Archaea, including Lokiarchaeota belonging to Asgard Archaea, the postulated archaeal ancestor of Eukaryotes. Strikingly, aCPSF1-dependent archaeal transcription termination reported here exposes a similar 3′-cleavage mode as the eukaryotic RNA polymerase II termination, thus would shed lights on understanding the evolutionary linking between archaeal and eukaryotic termination machineries.

scholarly journals aCPSF1 controlled archaeal transcription termination: a prototypical eukaryotic model

2019 ◽  
Author(s):  
Lei Yue ◽  
Jie Li ◽  
Bing Zhang ◽  
Lei Qi ◽  
Fangqing Zhao ◽  
...  
Keyword(s):  
Transcription Termination ◽  
Super Resolution ◽  
Microscopy Imaging ◽  
Genome Wide ◽  
Transcription Termination Factor ◽  
A Genome ◽  
Cold Sensitive ◽  
Archaeal Transcription ◽  
Rnap Ii

AbstractTranscription termination defines RNA 3′-ends and guarantees programmed transcriptomes, thus is an essential biological process for life. However, transcription termination mechanisms remain almost unknown in Archaea. Here reported the first general transcription termination factor of Archaea, the conserved ribonuclease aCPSF1, and elucidated its 3′-end cleavage dependent termination mechanism. Depletion of Mmp-aCPSF1 in a methanoarchaeon Methanococcus maripaludis caused a genome-wide transcription termination defect and overall transcriptome chaos, and cold-sensitive growth. Transcript-3′end-sequencing (Term-seq) revealed transcriptions mostly terminated downstream of a uridine-rich terminator motif, where Mmp-aCPSF1 performed cleavage. The endoribonuclease activity was determined essential to terminate transcription in vivo as well. Through super-resolution photoactivated localization microscopy imaging, co-immunoprecipitation, and chromatin immunoprecipitation, we demonstrated that Mmp-aCPSF1 localizes within nucleoid and associates with RNAP and chromosomes. aCPSF1 appears to co-evolve with archaeal RNAPs, and two distant orthologs each from Lokiarchaeota and Thaumarchaeota could replace Mmp-aCPSF1 to termination transcription. Thus, aCPSF1 dependent termination mechanism could be universally employed in Archaea, including Lokiarchaeota, one supposed archaeal ancestor of Eukaryotes. Therefore, the reported aCPSF1 cleavage-dependent termination mode not only hints an archetype of Eukaryotic 3′-end processing/cleavage triggered RNAP II termination, but also would shed lights on understanding the complex eukaryotic termination based on the simplified archaeal model.

scholarly journals The nucleosome DNA entry-exit site is important for transcription termination in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
A. Elizabeth Hildreth ◽  
Mitchell A. Ellison ◽  
Alex M. Francette ◽  
Julia M. Seraly ◽  
Lauren M. Lotka ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nucleosome Occupancy ◽  
Transcription Termination ◽  
Antisense Transcription ◽  
Exit Site ◽  
A Genome ◽  
Sense Strand ◽  
Native Sequence

AbstractCompared to other stages in the RNA polymerase II transcription cycle, the role of chromatin in transcription termination is poorly understood. Through a genetic screen, we identified histone mutant strains that exhibit transcriptional readthrough of terminators in vivo. Amino acid subtitutions map to the nucleosome DNA entry-exit site. On a genome-wide scale, the strongest H3 mutants revealed increased sense-strand transcription upstream and downstream of Pol II transcribed genes, increased antisense transcription overlapping gene bodies, and reduced nucleosome occupancy particularly at the 3’ ends of genes. Replacement of the native sequence downstream of a gene with a sequence that increases nucleosome occupancy in vivo reduced readthrough transcription and suppressed the effect of a DNA entry-exit site substitution. Our results suggest that nucleosomes can facilitate termination by serving as a barrier to RNA polymerase II progression and highlight the importance of the DNA entry-exit site in maintaining the integrity of the transcriptome.

scholarly journals CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

2007 ◽  
Vol 27 (5) ◽  
pp. 1631-1648 ◽  
Author(s):  
Igor Chernukhin ◽  
Shaharum Shamsuddin ◽  
Sung Yun Kang ◽  
Rosita Bergström ◽  
Yoo-Wook Kwon ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Activation ◽  
Ctcf Binding ◽  
Pol Ii ◽  
Genome Wide ◽  
Target Sites ◽  
On Chip

ABSTRACT CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. “Serial” chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

scholarly journals GTP-dependent Binding and Nuclear Transport of RNA Polymerase II by Npa3 Protein

2011 ◽  
Vol 286 (41) ◽  
pp. 35553-35561 ◽  
Author(s):  
Lidija Staresincic ◽  
Jane Walker ◽  
A. Barbara Dirac-Svejstrup ◽  
Richard Mitter ◽  
Jesper Q. Svejstrup
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Import ◽  
Chromatin Immunoprecipitation ◽  
Nuclear Transport ◽  
Proteomic Screen ◽  
Genome Wide ◽  
Importin Α

We identified XAB1 in a proteomic screen for factors that interact with human RNA polymerase II (RNAPII). Because XAB1 has a conserved Saccharomyces cerevisiae homologue called Npa3, yeast genetics and biochemical analysis were used to dissect the significance of the interaction. Degron-dependent Npa3 depletion resulted in genome-wide transcription decreases, correlating with a loss of RNAPII from genes as measured by chromatin immunoprecipitation. Surprisingly, however, transcription in vitro was unaffected by Npa3, suggesting that it affects a process that is not required for transcription in yeast extracts. Indeed, Npa3 depletion in vivo affects nuclear localization of RNAPII; the polymerase accumulates in the cytoplasm. Npa3 is a member of the GPN-LOOP family of GTPases. Npa3 mutants that either cannot bind GTP or that bind but cannot hydrolyze it are inviable and unable to support nuclear transport of RNAPII. Surprisingly, we were unable to detect interactions between Npa3 and proteins in the classical importin α/β pathway for nuclear import. Interestingly, Npa3-RNAPII binding is significantly increased by the addition of GTP or its slowly hydrolyzable analogue guanosine 5′-3-O-(thio)triphosphate (GTPγS). Moreover, the Npa3 mutant that binds GTP, but cannot hydrolyze it, binds RNAPII even in the absence of added GTP, whereas the mutant that cannot bind GTP is unable to bind the polymerase. Together, our data suggest that Npa3 defines an unconventional pathway for nuclear import of RNAPII, which involves GTP-dependent binding of Npa3 to the polymerase.

scholarly journals Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Anand Ranjan ◽  
Vu Q Nguyen ◽  
Sheng Liu ◽  
Jan Wisniewski ◽  
Jee Min Kim ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Transcription Initiation ◽  
General Mechanism ◽  
Pol Ii ◽  
Promoter Escape ◽  
Genome Wide ◽  
A Genome ◽  
Particle Imaging

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

scholarly journals Elongin A regulates transcription in vivo through enhanced RNA polymerase processivity

2020 ◽  
pp. jbc.RA120.015876
Author(s):  
Yating Wang ◽  
Liming Hou ◽  
M. Behfar Ardehali ◽  
Robert E. Kingston ◽  
Brian D Dynlacht
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Elongation ◽  
Rna Seq ◽  
Transcription Profiles ◽  
Genome Wide ◽  
The Impact

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. Our results suggest that Elongin A localizes to actively transcribed regions and potential enhancers, and the level of recruitment correlated with transcription levels. We also identified a large group of factors involved in transcription as Elongin A-associated factors. In addition, we found that loss of Elongin A leads to dramatically reduced levels of Ser2-phosphorylated, but not total, RNAPII, and cells depleted of Elongin A show stronger promoter RNAPII pausing, suggesting that Elongin A may be involved in the release of paused RNAPII. Our RNA-seq studies suggest that loss of Elongin A did not alter global transcription, and unlike prior in vitro studies, we did not observe a dramatic impact on RNAPII elongation rates in our cell-based nascent RNA-seq experiments upon Elongin A depletion. Taken together, our studies provide the first comprehensive analysis of the role of Elongin A in regulating transcription in vivo. Our studies also revealed that unlike prior in vitro findings, depletion of Elongin A has little impact on global transcription profiles and transcription elongation in vivo.

scholarly journals Elevated pausing of RNA Polymerase II underlies acquired resistance to ionizing radiation

2021 ◽  
Author(s):  
Kai Yuan ◽  
Honglu Liu ◽  
Chunhong Yu ◽  
Na Zhang ◽  
Yang Meng ◽  
...  
Keyword(s):  
Ionizing Radiation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Acquired Resistance ◽  
Cellular Processes ◽  
Genome Wide ◽  
A Genome ◽  
Xenograft Models ◽  
Resistance To Ionizing Radiation ◽  
Transcription Cycle

Abstract As the mainstay modality for many malignancies, ionizing radiation (IR) induces a variety of lesions in genomic DNA, evoking a multipronged DNA damage response to interrupt many cellular processes including transcription. How the global transcription cycle is altered by IR and whether it is contributing to the development of IR-resistance remain unaddressed. Here we report a genome-wide accumulation of paused RNA Polymerase II (RNAPII) after IR exposure. This increased pausing is partially maintained in cells acquired IR-resistance, notably on genes involved in radiation response and cell cycle, often leading to their downregulation. Individual knockdown some of these genes such as TP53 and NEK7 endows IR-sensitive cells with varying degrees of resistance, highlighting a novel link between elevated RNAPII pausing and the acquisition of IR-resistance. Accordingly, tuning-down the RNAPII pausing level by inhibiting CDK7 reverses IR-resistance both in cell culture and xenograft models. Our results suggest that modulation of the transcription cycle is a promising strategy to increase IR-sensitivity and thwart resistance.

Intrinsic sites of transcription termination and pausing in the c-myc gene

1988 ◽  
Vol 8 (10) ◽  
pp. 4389-4394
Author(s):  
T K Kerppola ◽  
C M Kane
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Xenopus Oocytes ◽  
Transcription Termination ◽  
Transcription Elongation ◽  
Rna Polymerase Iii ◽  
Intron 1 ◽  
Exon 1 ◽  
Myc Gene

We have studied transcription elongation and termination in the human c-myc gene. Transcription of c-myc gene sequences with purified mammalian RNA polymerase II revealed several sites of transcription termination and pausing in the vicinity of the exon 1-intron 1 junction. This region previously has been shown to block transcription elongation in vivo by nuclear run-on analysis (D. Bentley and M. Groudine, Nature [London] 321:702-706, 1986). These sites were recognized by purified RNA polymerase II, and we therefore designated them intrinsic sites of termination and pausing. Two of these sites cause termination of RNA polymerase III transcription as well. RNA polymerase II terminated transcription in a cluster of seven consecutive T residues in the nontranscribed strand and paused during transcription at three additional sites in this region. The intrinsic sites of transcription termination and pausing described here correspond closely to the 3' ends of transcripts synthesized in Xenopus oocytes injected with plasmids containing the c-myc termination region (D. Bentley and M. Groudine, Cell 53:245-256, 1988). This correspondence suggests that the intrinsic recognition of these termination and pause sites by purified RNA polymerase II may play a role in the transcription elongation block observed in vivo.

Termination and pausing of RNA polymerase II downstream of yeast polyadenylation sites

1993 ◽  
Vol 13 (9) ◽  
pp. 5159-5167
Author(s):  
L E Hyman ◽  
C L Moore
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Termination ◽  
In Vitro Synthesis ◽  
Polyadenylation Sites ◽  
Polymerase Pausing ◽  
A Site ◽  
Pause Site

Little is known about the transcriptional events which occur downstream of polyadenylation sites. Although the polyadenylation site of a gene can be easily identified, it has been difficult to determine the site of transcription termination in vivo because of the rapid processing of pre-mRNAs. Using an in vitro approach, we have shown that sequences from the 3' ends of two different Saccharomyces cerevisiae genes, ADH2 and GAL7, direct transcription termination and/or polymerase pausing in yeast nuclear extracts. In the case of the ADH2 sequence, the RNA synthesized in vitro ends approximately 50 to 150 nucleotides downstream of the poly(A) site. This RNA is not polyadenylated and may represent the primary transcript. A similarly sized nonpolyadenylated [poly(A)-] transcript can be detected in vivo from the same transcriptional template. A GAL7 template also directs the in vitro synthesis of an RNA which extends a short distance past the poly(A) site. However, a significant amount of the GAL7 RNA is polyadenylated at or close to the in vivo poly(A) site. Mutations of GAL7 or ADH2 poly(A) signals prevent polyadenylation but do not affect the in vitro synthesis of the extended poly(A)- transcript. Since transcription of the mutant template continues through this region in vivo, it is likely that a strong RNA polymerase II pause site lies within the 3'-end sequences. Our data support the hypothesis that the coupling of this pause site to a functional polyadenylation signal results in transcription termination.

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