Correlation in isolated nuclei of template-engaged RNA polymerase II, ovalbumin mRNA synthesis and estrogen receptor concentrations

Biochemistry ◽  
1985 ◽  
Vol 24 (6) ◽  
pp. 1275-1280 ◽  
Author(s):  
Robert N. Taylor ◽  
Roy G. Smith
Keyword(s):  
Estrogen Receptor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mrna Synthesis ◽  
Isolated Nuclei

scholarly journals Rpb4 Subunit Functions Mainly in mRNA Synthesis by RNA Polymerase II

2014 ◽  
Vol 289 (25) ◽  
pp. 17446-17452 ◽  
Author(s):  
Daniel Schulz ◽  
Nicole Pirkl ◽  
Elisabeth Lehmann ◽  
Patrick Cramer
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mrna Synthesis

scholarly journals Elongin C Contributes to RNA Polymerase II Degradation by the Interferon Antagonist NSs of La Crosse Orthobunyavirus

2020 ◽  
Vol 94 (7) ◽  
Author(s):  
Andreas Schoen ◽  
Simone Lau ◽  
Paul Verbruggen ◽  
Friedemann Weber
Keyword(s):  
United States ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ubiquitin Ligase ◽  
Nonstructural Protein ◽  
E3 Ubiquitin Ligase ◽  
The United States ◽  
Type I ◽  
La Crosse Virus ◽  
Mrna Synthesis

ABSTRACT Mosquito-borne La Crosse virus (LACV; genus Orthobunyavirus, family Peribunyaviridae, order Bunyavirales) causes up to 100 annual cases of severe meningoencephalitis in children and young adults in the United States. A major virulence factor of LACV is the nonstructural protein NSs, which inhibits host cell mRNA synthesis to prevent the induction of antiviral type I interferons (IFN-α/β). To achieve this host transcriptional shutoff, LACV NSs drives the proteasomal degradation of RPB1, the large subunit of mammalian RNA polymerase II. Here, we show that NSs acts in a surprisingly rapid manner, as RPB1 degradation was commencing already at 1 h postinfection. The RPB1 degradation was partially dependent on the cellular E3 ubiquitin ligase subunit Elongin C. Consequently, removal of Elongin C, but also of the subunits Elongin A or B by siRNA transfection partially rescued general RNAP II transcription and IFN-beta mRNA synthesis from the blockade by NSs. In line with these results, LACV NSs was found to trigger the redistribution of Elongin C out of nucleolar speckles, which, however, is an epiphenomenon rather than part of the NSs mechanism. Our study also shows that the molecular phenotype of LACV NSs is different from RNA polymerase II inhibitors like α-amanitin or Rift Valley fever virus NSs, indicating that LACV is unique in involving the Elongin complex to shut off host transcription and IFN response. IMPORTANCE The mosquito-borne La Crosse virus (LACV; genus Orthobunyavirus, family Peribunyaviridae, order Bunyavirales) is prevalent in the United States and can cause severe childhood meningoencephalitis. Its main virulence factor, the nonstructural protein NSs, is a strong inhibitor of the antiviral type I interferon (IFN) system. NSs acts by imposing a global host mRNA synthesis shutoff, mediated by NSs-driven proteasomal degradation of the RPB1 subunit of RNA polymerase II. Here, we show that RPB1 degradation commences as early as 1 h postinfection, and identify the E3 ubiquitin ligase subunit Elongin C (and its binding partners Elongins A and B) as an NSs cofactor involved in RPB1 degradation and in suppression of global as well as IFN-related mRNA synthesis.

scholarly journals An N-Terminal Region of Lassa Virus L Protein Plays a Critical Role in Transcription but Not Replication of the Virus Genome

2009 ◽  
Vol 84 (4) ◽  
pp. 1934-1944 ◽  
Author(s):  
Michaela Lelke ◽  
Linda Brunotte ◽  
Carola Busch ◽  
Stephan Günther
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Scale ◽  
Critical Role ◽  
Terminal Region ◽  
Lassa Virus ◽  
Mrna Synthesis ◽  
L Protein ◽  
Mrna Pool ◽  
Capping Enzymes

ABSTRACT The central domain of the 200-kDa Lassa virus L protein is a putative RNA-dependent RNA polymerase. N- and C-terminal domains may harbor enzymatic functions important for viral mRNA synthesis, including capping enzymes or cap-snatching endoribonucleases. In the present study, we have employed a large-scale mutagenesis approach to map functionally relevant residues in these regions. The main targets were acidic (Asp and Glu) and basic residues (Lys and Arg) known to form catalytic and binding sites of capping enzymes and endoribonucleases. A total of 149 different mutants were generated and tested in the Lassa virus replicon system. Nearly 25% of evolutionarily highly conserved acidic and basic side chains were dispensable for function of L protein in the replicon context. The vast majority of the remaining mutants had defects in both transcription and replication. Seven residues (Asp-89, Glu-102, Asp-119, Lys-122, Asp-129, Glu-180, and Arg-185) were selectively important for mRNA synthesis. The phenotype was particularly pronounced for Asp-89, Glu-102, and Asp-129, which were indispensable for transcription but could be replaced by a variety of amino acid residues without affecting genome replication. Bioinformatics disclosed the remote similarity of this region to type IIs endonucleases. The mutagenesis was complemented by experiments with the RNA polymerase II inhibitor α-amanitin, demonstrating dependence of viral transcription from the cellular mRNA pool. In conclusion, this paper describes an N-terminal region in L protein being important for mRNA, but not genome synthesis. Bioinformatics and cell biological experiments lend support to the hypothesis that this region could be part of a cap-snatching enzyme.

Transcription boundaries of U1 small nuclear RNA

1985 ◽  
Vol 5 (9) ◽  
pp. 2332-2340
Author(s):  
G R Kunkel ◽  
T Pederson
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Small Nuclear Rna ◽  
Cell Nuclei ◽  
Sm Proteins ◽  
Isolated Nuclei ◽  
Nuclear Rna ◽  
Rna Biosynthesis ◽  
Labeled Rna ◽  
Alpha Amanitin

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly.

scholarly journals Numerous post-translational modifications of RNA polymerase II subunit Rpb4 link transcription to post-transcriptional mechanisms

2019 ◽  
Author(s):  
Stephen Richard ◽  
Lital Gross ◽  
Jonathan Fischer ◽  
Keren Bendalak ◽  
Tamar Ziv ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
The Other ◽  
Mrna Synthesis ◽  
Mutant Type ◽  
Post Translational Modifications ◽  
Pol Ii ◽  
One Stage

AbstractRpb4/7 binds RNA Polymerase II (Pol II) transcripts co-transcriptionally and accompanies them throughout their lives. By virtue of its capacity to interact with key regulators (e.g., Pol II, eIF3, Pat1) both temporarily and spatially, Rpb4/7 regulates the major stages of the mRNA lifecycle. Here we show that Rpb4/7 can undergo over 100 combinations of post-translational modifications (PTMs). Remarkably, the Rpb4/7 PTMs repertoire changes as the mRNA/Rpb4/7 complex progresses from one stage to the next. A mutagenesis approach in residues that undergo PTMs suggests that temporal Rpb4 PTMs regulate its interactions with key regulators of gene expression that control transcriptional and post-transcriptional stages. Moreover, one mutant type specifically affects mRNA synthesis despite its normal association with Pol II, whereas the other affects both mRNA synthesis and decay; both types disrupt the balance between mRNA synthesis and decay (‘mRNA buffering’) and the cell’s capacity to respond to the environment. Taken together, we propose that temporal Rpb4/7 PTMs are involved in cross talks among the various stages of the mRNA lifecycle.

scholarly journals Estrogen-induced modification of uterine RNA polymerase activity depends on localization of the estrogen receptor

2007 ◽  
Vol 59 (2) ◽  
pp. 105-112
Author(s):  
Zorica Zakula ◽  
Esma Isenovic ◽  
Mojca Stojiljkovic ◽  
G. Koricanac ◽  
Snezana Tepavcevic ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Polymerase Activity ◽  
Rna Polymerase I ◽  
Female Rats ◽  
Cytosol Fraction ◽  
Ovx Rats ◽  
Rna Polymerase Activity ◽  
Polymerase I

The aim of this study was to examine the effects of estradiol (E2) on activity of RNA polymerase I and RNA polymerase II in uterine nuclei of ovariectomized (OVX) female rats. The obtained results show that estrogen-receptor (E-R) complexes in 30 min induced an increase of polymerase II activity. A second increase of polymerase II activity was observed after 3 h-incubation of nuclei with the E-R complex formed in the cytosol fraction. However, activity of polymerase I was increased 2 h after the start of incubation, with highest activity detected at 3 h in nuclei incubated with E-R complexes. On the contrary, no stimulatory effect on either polymerase I or polymerase II activity was observed in nuclei incubated with E2 alone. These results indicate that E2 stimulates the cytosolic estrogen receptor (ER), which in turn causes uterotrophic responses in OVX rats. In addition, they suggest that in order to provoke uterotrophic responses E-R complexes formed in the cytosol need to be retained in the nucleus for a longer period of time. .

scholarly journals Transcription boundaries of U1 small nuclear RNA.

1985 ◽  
Vol 5 (9) ◽  
pp. 2332-2340 ◽  
Author(s):  
G R Kunkel ◽  
T Pederson
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Small Nuclear Rna ◽  
Cell Nuclei ◽  
Sm Proteins ◽  
Isolated Nuclei ◽  
Nuclear Rna ◽  
Rna Biosynthesis ◽  
Labeled Rna ◽  
Alpha Amanitin

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly.

scholarly journals Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis.

1987 ◽  
Vol 7 (5) ◽  
pp. 1602-1611 ◽  
Author(s):  
M Nonet ◽  
C Scafe ◽  
J Sexton ◽  
R Young
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Direct Evidence ◽  
Mrna Synthesis ◽  
Wild Type ◽  
Gene Encoding ◽  
Conditional Mutant ◽  
Defective Rna

We have isolated a yeast conditional mutant which rapidly ceases synthesis of mRNA when subjected to the nonpermissive temperature. This mutant (rpb1-1) was constructed by replacing the wild-type chromosomal copy of the gene encoding the largest subunit of RNA polymerase II with one mutagenized in vitro. The rapid cessation of mRNA synthesis in vivo and the lack of RNA polymerase II activity in crude extracts indicate that the mutant possesses a functionally defective, rather than an assembly-defective, RNA polymerase II. The shutdown in mRNA synthesis in the rpb1-1 mutant has pleiotropic effects on the synthesis of other RNAs and on the heat shock response. This mutant provides direct evidence that the RPB1 protein has a functional role in mRNA synthesis.

scholarly journals The yeast exoribonuclease Xrn1 and associated factors modulate RNA polymerase II processivity in 5' and 3' gene regions

2020 ◽  
Vol 295 (33) ◽  
pp. 11435-11454 ◽  
Author(s):  
Jonathan Fischer ◽  
Yun S. Song ◽  
Nir Yosef ◽  
Julia di Iulio ◽  
L. Stirling Churchman ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Mrna Decay ◽  
Indirect Evidence ◽  
Yeast Cells ◽  
Mrna Levels ◽  
Mrna Synthesis ◽  
Pol Ii ◽  
Polyadenylation Sites

mRNA levels are determined by the balance between mRNA synthesis and decay. Protein factors that mediate both processes, including the 5'-3' exonuclease Xrn1, are responsible for a cross-talk between the two processes that buffers steady-state mRNA levels. However, the roles of these proteins in transcription remain elusive and controversial. Applying native elongating transcript sequencing (NET-seq) to yeast cells, we show that Xrn1 functions mainly as a transcriptional activator and that its disruption manifests as a reduction of RNA polymerase II (Pol II) occupancy downstream of transcription start sites. By combining our sequencing data and mathematical modeling of transcription, we found that Xrn1 modulates transcription initiation and elongation of its target genes. Furthermore, Pol II occupancy markedly increased near cleavage and polyadenylation sites in xrn1Δ cells, whereas its activity decreased, a characteristic feature of backtracked Pol II. We also provide indirect evidence that Xrn1 is involved in transcription termination downstream of polyadenylation sites. We noted that two additional decay factors, Dhh1 and Lsm1, seem to function similarly to Xrn1 in transcription, perhaps as a complex, and that the decay factors Ccr4 and Rpb4 also perturb transcription in other ways. Interestingly, the decay factors could differentiate between SAGA- and TFIID-dominated promoters. These two classes of genes responded differently to XRN1 deletion in mRNA synthesis and were differentially regulated by mRNA decay pathways, raising the possibility that one distinction between these two gene classes lies in the mechanisms that balance mRNA synthesis with mRNA decay.

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