Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast

Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3’ untranslated region in order to regulate mRNA function. Here, we present a systematic analysis of 3’ end formation factors, which revealed 3’UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3’UTRs. We show that the anti-cancer drug cordycepin, 3’ deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin-mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an rpb1 mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3’ end processing and controlled by the availability of 3’ end factors, nucleotide levels and chromatin landscape.

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Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast

10.1101/2020.12.01.407171 ◽

2020 ◽

Author(s):

Rachael E. Turner ◽

Paul F. Harrison ◽

Angavai Swaminathan ◽

Calvin A. Kraupner-Taylor ◽

Melissa J. Curtis ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Alternative Polyadenylation ◽

Cancer Drug ◽

Systematic Analysis ◽

The Core ◽

Anti Cancer ◽

Chromatin Template ◽

Eukaryotic Mrnas ◽

Rnap Ii ◽

Alternative Sites

ABSTRACTMost eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3’ untranslated region in order to regulate mRNA function. Here we present a systematic analysis of 3’ end formation factors, which revealed 3’UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3’UTRs. We show that the anti-cancer drug cordycepin, 3’ deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an rpb1 mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3’ end processing and controlled by the availability of 3’ end factors, nucleotide levels and chromatin landscape.

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Regulation of alternative polyadenylation in the yeast Saccharomyces cerevisiae by histone H3K4 and H3K36 methyltransferases

Nucleic Acids Research ◽

10.1093/nar/gkaa292 ◽

2020 ◽

Vol 48 (10) ◽

pp. 5407-5425 ◽

Cited By ~ 1

Author(s):

Katarzyna Kaczmarek Michaels ◽

Salwa Mohd Mostafa ◽

Julia Ruiz Capella ◽

Claire L Moore

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase Ii ◽

Nucleosome Occupancy ◽

Alternative Polyadenylation ◽

Nutritional Stress ◽

Yeast Saccharomyces Cerevisiae ◽

Terminal Domain ◽

Histone H3k4 ◽

Rnap Ii ◽

And Control

Abstract Adjusting DNA structure via epigenetic modifications, and altering polyadenylation (pA) sites at which precursor mRNA is cleaved and polyadenylated, allows cells to quickly respond to environmental stress. Since polyadenylation occurs co-transcriptionally, and specific patterns of nucleosome positioning and chromatin modifications correlate with pA site usage, epigenetic factors potentially affect alternative polyadenylation (APA). We report that the histone H3K4 methyltransferase Set1, and the histone H3K36 methyltransferase Set2, control choice of pA site in Saccharomyces cerevisiae, a powerful model for studying evolutionarily conserved eukaryotic processes. Deletion of SET1 or SET2 causes an increase in serine-2 phosphorylation within the C-terminal domain of RNA polymerase II (RNAP II) and in the recruitment of the cleavage/polyadenylation complex, both of which could cause the observed switch in pA site usage. Chemical inhibition of TOR signaling, which causes nutritional stress, results in Set1- and Set2-dependent APA. In addition, Set1 and Set2 decrease efficiency of using single pA sites, and control nucleosome occupancy around pA sites. Overall, our study suggests that the methyltransferases Set1 and Set2 regulate APA induced by nutritional stress, affect the RNAP II C-terminal domain phosphorylation at Ser2, and control recruitment of the 3′ end processing machinery to the vicinity of pA sites.

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Core promoter-selective RNA polymerase II transcription

Biochemical Society Symposium ◽

10.1042/bss0730225 ◽

2006 ◽

Vol 73 ◽

pp. 225-236 ◽

Cited By ~ 29

Author(s):

Petra Gross ◽

Thomas Oelgeschläger

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Core Promoter ◽

Promoter Sequence ◽

Tata Box ◽

Promoter Elements ◽

The Core ◽

Core Promoter Sequence ◽

Rnap Ii

The initiation of mRNA synthesis in eukaryotic cells is a complex and highly regulated process that requires the assembly of general transcription factors and RNAP II (RNA polymerase II; also abbreviated as Pol II) into a pre-initiation complex at the core promoter. The core promoter is defined as the minimal DNA region that is sufficient to direct low levels of activator-independent (basal) transcription by RNAP II in vitro. The core promoter typically extends approx. 40 bp up- and down-stream of the start site of transcription and can contain several distinct core promoter sequence elements. Core promoters in higher eukaryotes are highly diverse in structure, and each core promoter sequence element is only found in a subset of genes. So far, only TATA box and INR (initiator) element have been shown to be capable of directing accurate RNAP II transcription initiation independent of other core promoter elements. Computational analysis of metazoan genomes suggests that the prevalence of the TATA box has been overestimated in the past and that the majority of human genes are TATA-less. While TATA-mediated transcription initiation has been studied in great detail and is very well understood, very little is known about the factors and mechanisms involved in the function of the INR and other core promoter elements. Here we summarize our current understanding of the factors and mechanisms involved in core promoter-selective transcription and discuss possible pathways through which diversity in core promoter architecture might contribute to combinatorial gene regulation in metazoan cells.

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The anti-cancer drug camptothecin inhibits elongation but stimulates initiation of RNA polymerase II transcription

Carcinogenesis ◽

10.1093/carcin/17.1.31 ◽

1996 ◽

Vol 17 (1) ◽

pp. 31-36 ◽

Cited By ~ 41

Author(s):

Mats Ljungman ◽

Philip C. Hanawalt

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Cancer Drug ◽

Anti Cancer ◽

Rna Polymerase Ii Transcription

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Ibulocydine Is a Novel Prodrug Cdk Inhibitor That Effectively Induces Apoptosis in Hepatocellular Carcinoma Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m110.209551 ◽

2011 ◽

Vol 286 (22) ◽

pp. 19662-19671 ◽

Cited By ~ 24

Author(s):

Seung-Ju Cho ◽

Young-Jong Kim ◽

Young-Joon Surh ◽

B. Moon Kim ◽

Seung-Ki Lee

Keyword(s):

Hepatocellular Carcinoma ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Large Subunit ◽

Cancer Drug ◽

Cdk Inhibitors ◽

Cdk Inhibitor ◽

Anti Cancer ◽

Induced Apoptosis ◽

Hcc Cells

Hepatocellular carcinoma (HCC) is frequently associated with abnormalities in cell cycle regulation, leading to increased activity of cyclin-dependent kinases (Cdks) due to the loss, or low expression of, Cdk inhibitors. In this study, we showed that ibulocydine (an isobutyrate prodrug of the specific Cdk inhibitor, BMK-Y101) is a candidate anti-cancer drug for HCC. Ibulocydine has high activity against Cdk7/cyclin H/Mat1 and Cdk9/cyclin T. Ibulocydine inhibited the growth of HCC cells more effectively than other Cdk inhibitors, including olomoucine and roscovitine, whereas ibulocydine as well as the other Cdk inhibitors and BMK-Y101 minimally influenced the growth of normal hepatocyte cells. Ibulocydine induced apoptosis in HCC cells, most likely by inhibiting Cdk7 and Cdk9. In vitro treatment of HCC cells with ibulocydine rapidly blocked phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II, a process mediated by Cdk7/9. Anti-apoptotic gene products such as Mcl-1, survivin, and X-linked IAP (XIAP) are crucial for the survival of many cell types, including HCC. Following the inhibition of RNA polymerase II phosphorylation, ibulocydine caused rapid down-regulation of Mcl-1, survivin, and XIAP, thus inducing apoptosis. Furthermore, ibulocydine effectively induced apoptosis in HCC xenografts with no toxic side effects. These results suggest that ibulocydine is a strong candidate anti-cancer drug for the treatment of HCC.

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Core Promoter Elements and TAFs Contribute to the Diversity of Transcriptional Activation in Vertebrates

Molecular and Cellular Biology ◽

10.1128/mcb.23.20.7350-7362.2003 ◽

2003 ◽

Vol 23 (20) ◽

pp. 7350-7362 ◽

Cited By ~ 18

Author(s):

Zheng Chen ◽

James L. Manley

Keyword(s):

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Core Promoter ◽

Simian Virus 40 ◽

Simian Virus ◽

Promoter Strength ◽

Promoter Elements ◽

The Core ◽

Activation Mechanisms ◽

Rnap Ii

ABSTRACT Gene-specific transcriptional activation is a multistep process that requires numerous protein factors and DNA elements, including enhancers and the core promoter. To investigate the roles of core promoter elements in transcriptional activation in vertebrates, we examined expression and factor occupancy on representative promoters in chicken DT40 cells containing a conditional TATA binding protein (TBP)-associated factor 9 allele (TAF9). Characterized core elements, including TATA box-flanking regions and the downstream promoter element, were found to play significant roles in determining promoter strength, response to activators, and factor occupancy and recruitment. The requirement for TAF9 was found to be highly promoter specific, and TAF9 dependence and promoter occupancy were not always correlated. We also describe contrasting examples of factor recruitment and activation mechanisms at different promoters, highlighted by the nearly opposite mechanisms utilized by the simian virus 40 enhancer and p53. With the core promoters analyzed, the former functions by facilitating RNA polymerase II (RNAP II) recruitment to a preassembled TBP/TFIIB-containing scaffold and p53 strongly recruits TBP and TFIIB while RNAP II levels remain modest. Taken together, our results illustrate both the important roles of core promoter elements and the remarkable diversity that characterizes transcriptional activation mechanisms in vertebrates.

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