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1.
Genes Dev. 2018 Feb 1;32(3-4):297-308. doi: 10.1101/gad.310896.117. Epub 2018 Feb 26.

Transcription elongation rate affects nascent histone pre-mRNA folding and 3' end processing.

Author information

1
Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora, Colorado 80045, USA.

Abstract

Transcription elongation rate influences cotranscriptional pre-mRNA maturation, but how such kinetic coupling works is poorly understood. The formation of nonadenylated histone mRNA 3' ends requires recognition of an RNA structure by stem-loop-binding protein (SLBP). We report that slow transcription by mutant RNA polymerase II (Pol II) caused accumulation of polyadenylated histone mRNAs that extend past the stem-loop processing site. UV irradiation, which decelerates Pol II elongation, also induced long poly(A)+ histone transcripts. Inhibition of 3' processing by slow Pol II correlates with failure to recruit SLBP to histone genes. Chemical probing of nascent RNA structure showed that the stem-loop fails to fold in transcripts made by slow Pol II, thereby explaining the absence of SLBP and failure to process 3' ends. These results show that regulation of transcription speed can modulate pre-mRNA processing by changing nascent RNA structure and suggest a mechanism by which alternative processing could be controlled.

KEYWORDS:

cotranscriptional RNA folding; histone mRNA 3′ end processing; kinetic coupling; stem–loop binding protein; transcription elongation rate

PMID:
29483154
PMCID:
PMC5859970
[Available on 2018-08-01]
DOI:
10.1101/gad.310896.117
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2.
Cell Rep. 2017 Aug 1;20(5):1173-1186. doi: 10.1016/j.celrep.2017.07.021.

Human TFIIH Kinase CDK7 Regulates Transcription-Associated Chromatin Modifications.

Author information

1
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80303, USA; Department of Molecular, Cell, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
2
Department Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA.
3
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80303, USA.
4
BioFrontiers Institute, University of Colorado, Boulder, CO 80309, USA; Linda Crnic Institute for Down Syndrome, University of Colorado School of Medicine, Aurora, CO 80045, USA.
5
Department of Molecular, Cell, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
6
Department of Biochemistry & Molecular Genetics, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, USA.
7
Department of Molecular, Cell, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA; BioFrontiers Institute, University of Colorado, Boulder, CO 80309, USA; Linda Crnic Institute for Down Syndrome, University of Colorado School of Medicine, Aurora, CO 80045, USA.
8
Department of Molecular, Cell, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA; Linda Crnic Institute for Down Syndrome, University of Colorado School of Medicine, Aurora, CO 80045, USA.
9
Department Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA. Electronic address: david.bentley@ucdenver.edu.
10
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80303, USA. Electronic address: taatjes@colorado.edu.

Abstract

CDK7 phosphorylates the RNA polymerase II (pol II) C-terminal domain CTD and activates the P-TEFb-associated kinase CDK9, but its regulatory roles remain obscure. Here, using human CDK7 analog-sensitive (CDK7as) cells, we observed reduced capping enzyme recruitment, increased pol II promoter-proximal pausing, and defective termination at gene 3' ends upon CDK7 inhibition. We also noted that CDK7 regulates chromatin modifications downstream of transcription start sites. H3K4me3 spreading was restricted at gene 5' ends and H3K36me3 was displaced toward gene 3' ends in CDK7as cells. Mass spectrometry identified factors that bound TFIIH-phosphorylated versus P-TEFb-phosphorylated CTD (versus unmodified); capping enzymes and H3K4 methyltransferase complexes, SETD1A/B, selectively bound phosphorylated CTD, and the H3K36 methyltransferase SETD2 specifically bound P-TEFb-phosphorylated CTD. Moreover, TFIIH-phosphorylated CTD stimulated SETD1A/B activity toward nucleosomes, revealing a mechanistic basis for CDK7 regulation of H3K4me3 spreading. Collectively, these results implicate a CDK7-dependent "CTD code" that regulates chromatin marks in addition to RNA processing and pol II pausing.

KEYWORDS:

H3K36me3; H3K4me3; Mediator; P-TEFb; RNA-seq; TFIIH; THZ1; chromatin; epigenetic; proteomics

PMID:
28768201
PMCID:
PMC5564226
DOI:
10.1016/j.celrep.2017.07.021
[Indexed for MEDLINE]
Free PMC Article
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3.
Cancer Res. 2017 Sep 15;77(18):4934-4946. doi: 10.1158/0008-5472.CAN-16-3541. Epub 2017 Jul 20.

Breast Cancer Suppression by Progesterone Receptors Is Mediated by Their Modulation of Estrogen Receptors and RNA Polymerase III.

Author information

1
Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, Colorado. Jessica.Finlay-Schultz@ucdenver.edu Carol.Sartorius@ucdenver.edu.
2
RNA Bioscience Initiative, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
3
Department of Medicine, Division of Medical Oncology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
4
Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
5
Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Abstract

Greater than 50% of estrogen receptor (ER)-positive breast cancers coexpress the progesterone receptor (PR), which can directly and globally modify ER action to attenuate tumor growth. However, whether this attenuation is mediated only through PR-ER interaction remains unknown. To address this question, we assessed tumor growth in ER/PR-positive patient-derived xenograft models of breast cancer, where both natural and synthetic progestins were found to antagonize the mitogenic effects of estrogens. Probing the genome-wide mechanisms by which this occurs, we documented that chronic progestin treatment blunted ER-mediated gene expression up to 2-fold at the level of mRNA transcripts. Unexpectedly, <25% of all ER DNA binding events were affected by the same treatment. The PR cistrome displayed a bimodal distribution. In one group, >50% of PR binding sites were co-occupied by ER, with a propensity for both receptors to coordinately gain or lose binding in the presence of progesterone. In the second group, PR but not ER was associated with a large fraction of RNA polymerase III-transcribed tRNA genes, independent of hormone treatment. Notably, we discovered that PR physically associated with the Pol III holoenzyme. Select pre-tRNAs and mature tRNAs with PR and POLR3A colocalized at their promoters were relatively decreased in estrogen + progestin-treated tumors. Our results illuminate how PR may indirectly impede ER action by reducing the bioavailability of translational molecules needed for tumor growth. Cancer Res; 77(18); 4934-46. ©2017 AACR.

PMID:
28729413
PMCID:
PMC5600857
[Available on 2018-09-15]
DOI:
10.1158/0008-5472.CAN-16-3541
[Indexed for MEDLINE]
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4.
Mol Cell. 2017 May 18;66(4):546-557.e3. doi: 10.1016/j.molcel.2017.04.016. Epub 2017 May 11.

RNA Pol II Dynamics Modulate Co-transcriptional Chromatin Modification, CTD Phosphorylation, and Transcriptional Direction.

Author information

1
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, PO Box 6511, Aurora, CO 80045, USA.
2
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, PO Box 6511, Aurora, CO 80045, USA. Electronic address: david.bentley@ucdenver.edu.

Abstract

Eukaryotic genes are marked by conserved post-translational modifications on the RNA pol II C-terminal domain (CTD) and the chromatin template. How the 5'-3' profiles of these marks are established is poorly understood. Using pol II mutants in human cells, we found that slow transcription repositioned specific co-transcriptionally deposited chromatin modifications; histone H3 lysine 36 trimethyl (H3K36me3) shifted within genes toward 5' ends, and histone H3 lysine 4 dimethyl (H3K4me2) extended farther upstream of start sites. Slow transcription also evoked a hyperphosphorylation of CTD Ser2 residues at 5' ends of genes that is conserved in yeast. We propose a "dwell time in the target zone" model to explain the effects of transcriptional dynamics on the establishment of co-transcriptionally deposited protein modifications. Promoter-proximal Ser2 phosphorylation is associated with a longer pol II dwell time at start sites and reduced transcriptional polarity because of strongly enhanced divergent antisense transcription at promoters. These results demonstrate that pol II dynamics help govern the decision between sense and divergent antisense transcription.

KEYWORDS:

H3K4me2; K3K36me3; antisense transcription; bidirectional transcription; histone methylation; kinetic coupling; pol II CTD S2 phosphorylation; pol II dynamics; transcription elongation rate

PMID:
28506463
PMCID:
PMC5488731
[Available on 2018-05-18]
DOI:
10.1016/j.molcel.2017.04.016
[Indexed for MEDLINE]
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5.
J Mol Biol. 2016 Jun 19;428(12):2623-2635. doi: 10.1016/j.jmb.2016.04.017. Epub 2016 Apr 20.

Coupling of RNA Polymerase II Transcription Elongation with Pre-mRNA Splicing.

Author information

1
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, P.O. Box 6511, Aurora, CO 80045, USA.
2
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, P.O. Box 6511, Aurora, CO 80045, USA. Electronic address: david.bentley@ucdenver.edu.

Abstract

Pre-mRNA maturation frequently occurs at the same time and place as transcription by RNA polymerase II. The co-transcriptionality of mRNA processing has permitted the evolution of mechanisms that functionally couple transcription elongation with diverse events that occur on the nascent RNA. This review summarizes the current understanding of the relationship between transcriptional elongation through a chromatin template and co-transcriptional splicing including alternative splicing decisions that affect the expression of most human genes.

KEYWORDS:

CTD; RNA polymerase II; alternative splicing; kinetic coupling; transcription elongation

PMID:
27107644
PMCID:
PMC4893998
DOI:
10.1016/j.jmb.2016.04.017
[Indexed for MEDLINE]
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6.
Biotechniques. 2016 Feb 1;60(2):69-74. doi: 10.2144/000114378. eCollection 2016 Feb.

Selectable one-step PCR-mediated integration of a degron for rapid depletion of endogenous human proteins.

Author information

1
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO.

Abstract

Manipulation of protein stability with ligand-regulated degron fusions is a powerful method for investigating gene function. We developed a selectable cassette for easy C-terminal tagging of endogenous human proteins with the E. coli dihydrofolate reductase (eDHFR) degron using CRISPR/Cas9 genome editing. This cassette permits high-efficiency recovery of correct integration events using an in-frame self-cleaving 2A peptide and the puromycin resistance gene. PCR amplified donor eDHFR cassette fragments with 100 bases of homology on each end are integrated by homology-directed repair (HDR) of guide RNA (gRNA)-targeted double-stranded DNA breaks at the 3' ends of open reading frames (ORFs). As proof of principle, we generated cell lines in which three endogenous proteins were tagged with the eDHFR degron. When the antibiotic trimethoprim is removed from the media, each of the eDHFR-tagged proteins was depleted by >90% within 2-4 h, and this depletion was reversed by re-addition of trimethoprim. Since puromycin selection permits recovery of in-frame degron fusions with high efficiency using only 100-bp long regions of homology, this method should be applicable on a genome-wide scale for generating libraries of conditional mutant cell lines.

KEYWORDS:

CRISPR, homology directed repair; genome editing; human degron mutants; protein destabilization domain; regulated protein stability

PMID:
26842351
PMCID:
PMC4893940
DOI:
10.2144/000114378
[Indexed for MEDLINE]
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7.
Mol Cell Biol. 2015 Nov 2;36(2):295-303. doi: 10.1128/MCB.00898-15. Print 2016 Jan 15.

Coordination of RNA Polymerase II Pausing and 3' End Processing Factor Recruitment with Alternative Polyadenylation.

Author information

1
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado, USA.
2
Department of Microbiology, Immunology & Molecular Genetics, University of Kentucky Medical Center, Lexington, Kentucky, USA.
3
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado, USA david.bentley@ucdenver.edu.

Abstract

Most mammalian genes produce transcripts whose 3' ends are processed at multiple alternative positions by cleavage/polyadenylation (CPA). Poly(A) site cleavage frequently occurs cotranscriptionally and is facilitated by CPA factor binding to the RNA polymerase II (Pol II) C-terminal domain (CTD) phosphorylated on Ser2 residues of its heptad repeats (YS2PTSPS). The function of cotranscriptional events in the selection of alternative poly(A) sites is poorly understood. We investigated Pol II pausing, CTD Ser2 phosphorylation, and processing factor CstF recruitment at wild-type and mutant IgM transgenes that use alternative poly(A) sites to produce mRNAs encoding the secreted and membrane-bound forms of the immunoglobulin (Ig) heavy chain. The results show that the sites of Pol II pausing and processing factor recruitment change depending on which poly(A) site is utilized. In contrast, the extent of Pol II CTD Ser2 phosphorylation does not closely correlate with poly(A) site selection. We conclude that changes in properties of the transcription elongation complex closely correlate with utilization of different poly(A) sites, suggesting that cotranscriptional events may influence the decision between alternative modes of pre-mRNA 3' end processing.

PMID:
26527620
PMCID:
PMC4719304
DOI:
10.1128/MCB.00898-15
[Indexed for MEDLINE]
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8.
Mol Cell. 2015 Oct 15;60(2):256-67. doi: 10.1016/j.molcel.2015.09.026.

Effects of Transcription Elongation Rate and Xrn2 Exonuclease Activity on RNA Polymerase II Termination Suggest Widespread Kinetic Competition.

Author information

1
Deptartment of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, PO Box 6511, Aurora, CO 80045, USA.
2
Deptartment of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, PO Box 6511, Aurora, CO 80045, USA. Electronic address: david.bentley@ucdenver.edu.

Abstract

The torpedo model of transcription termination asserts that the exonuclease Xrn2 attacks the 5'PO4-end exposed by nascent RNA cleavage and chases down the RNA polymerase. We tested this mechanism using a dominant-negative human Xrn2 mutant and found that it delayed termination genome-wide. Xrn2 nuclease inactivation caused strong termination defects downstream of most poly(A) sites and modest delays at some histone and U snRNA genes, suggesting that the torpedo mechanism is not limited to poly(A) site-dependent termination. A central untested feature of the torpedo model is that there is kinetic competition between the exonuclease and the pol II elongation complex. Using pol II rate mutants, we found that slow transcription robustly shifts termination upstream, and fast elongation extends the zone of termination further downstream. These results suggest that kinetic competition between elongating pol II and the Xrn2 exonuclease is integral to termination of transcription on most human genes.

Comment in

PMID:
26474067
PMCID:
PMC4654110
DOI:
10.1016/j.molcel.2015.09.026
[Indexed for MEDLINE]
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10.
Genes Dev. 2014 Dec 1;28(23):2663-76. doi: 10.1101/gad.252106.114.

Pre-mRNA splicing is facilitated by an optimal RNA polymerase II elongation rate.

Author information

1
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045, USA;
2
Department of Cellular and Molecular Medicine, Institute of Genomic Medicine, University of California at San Diego, San Diego, California 92093, USA.
3
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045, USA; david.bentley@ucdenver.edu.

Abstract

Alternative splicing modulates expression of most human genes. The kinetic model of cotranscriptional splicing suggests that slow elongation expands and that fast elongation compresses the "window of opportunity" for recognition of upstream splice sites, thereby increasing or decreasing inclusion of alternative exons. We tested the model using RNA polymerase II mutants that change average elongation rates genome-wide. Slow and fast elongation affected constitutive and alternative splicing, frequently altering exon inclusion and intron retention in ways not predicted by the model. Cassette exons included by slow and excluded by fast elongation (type I) have weaker splice sites, shorter flanking introns, and distinct sequence motifs relative to "slow-excluded" and "fast-included" exons (type II). Many rate-sensitive exons are misspliced in tumors. Unexpectedly, slow and fast elongation often both increased or both decreased inclusion of a particular exon or retained intron. These results suggest that an optimal rate of transcriptional elongation is required for normal cotranscriptional pre-mRNA splicing.

KEYWORDS:

alternative splicing; cotranscriptional splicing; intron retention; kinetic coupling; transcription elongation rate

PMID:
25452276
PMCID:
PMC4248296
DOI:
10.1101/gad.252106.114
[Indexed for MEDLINE]
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11.
Nat Rev Genet. 2014 Mar;15(3):163-75. doi: 10.1038/nrg3662. Epub 2014 Feb 11.

Coupling mRNA processing with transcription in time and space.

Author information

1
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, MS8101, PO BOX 6511, Aurora, Colorado 80045, USA.

Abstract

Maturation of mRNA precursors often occurs simultaneously with their synthesis by RNA polymerase II (Pol II). The co-transcriptional nature of mRNA processing has permitted the evolution of coupling mechanisms that coordinate transcription with mRNA capping, splicing, editing and 3' end formation. Recent experiments using sophisticated new methods for analysis of nascent RNA have provided important insights into the relative amount of co-transcriptional and post-transcriptional processing, the relationship between mRNA elongation and processing, and the role of the Pol II carboxy-terminal domain (CTD) in regulating these processes.

PMID:
24514444
PMCID:
PMC4304646
DOI:
10.1038/nrg3662
[Indexed for MEDLINE]
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12.
Cell Rep. 2014 Jan 30;6(2):325-35. doi: 10.1016/j.celrep.2013.12.021. Epub 2014 Jan 9.

The histone-H3K4-specific demethylase KDM5B binds to its substrate and product through distinct PHD fingers.

Author information

1
Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA.
2
Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
3
Dan L. Duncan Cancer Center, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
4
Basic Science Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
5
Department of Biochemistry and Biophysics and the Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
6
Genes and Development Graduate Program, The University of Texas Graduate School of Biomedical Sciences, Houston, TX 77030, USA.
7
Molecular Biology Program, University of Colorado School of Medicine, Aurora, CO 80045, USA.
8
Molecular Biology Program, University of Colorado School of Medicine, Aurora, CO 80045, USA; Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA.
9
Basic Science Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Department of Radiation Oncology, University Washington School of Medicine, Seattle, WA 98109, USA.
10
Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Genes and Development Graduate Program, The University of Texas Graduate School of Biomedical Sciences, Houston, TX 77030, USA; Center for Cancer Epigenetics and Center for Stem Cell and Developmental Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. Electronic address: xbshi@mdanderson.org.
11
Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA; Molecular Biology Program, University of Colorado School of Medicine, Aurora, CO 80045, USA. Electronic address: tatiana.kutateladze@ucdenver.edu.

Abstract

The histone lysine demethylase KDM5B regulates gene transcription and cell differentiation and is implicated in carcinogenesis. It contains multiple conserved chromatin-associated domains, including three PHD fingers of unknown function. Here, we show that the first and third, but not the second, PHD fingers of KDM5B possess histone binding activities. The PHD1 finger is highly specific for unmodified histone H3 (H3K4me0), whereas the PHD3 finger shows preference for the trimethylated histone mark H3K4me3. RNA-seq analysis indicates that KDM5B functions as a transcriptional repressor for genes involved in inflammatory responses, cell proliferation, adhesion, and migration. Biochemical analysis reveals that KDM5B associates with components of the nucleosome remodeling and deacetylase (NuRD) complex and may cooperate with the histone deacetylase 1 (HDAC1) in gene repression. KDM5B is downregulated in triple-negative breast cancer relative to estrogen-receptor-positive breast cancer. Overexpression of KDM5B in the MDA-MB 231 breast cancer cells suppresses cell migration and invasion, and the PHD1-H3K4me0 interaction is essential for inhibiting migration. These findings highlight tumor-suppressive functions of KDM5B in triple-negative breast cancer cells and suggest a multivalent mechanism for KDM5B-mediated transcriptional regulation.

PMID:
24412361
PMCID:
PMC3918441
DOI:
10.1016/j.celrep.2013.12.021
[Indexed for MEDLINE]
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13.
Genet Res Int. 2012;2012:170173. doi: 10.1155/2012/170173. Epub 2012 Jan 29.

Control of Transcriptional Elongation by RNA Polymerase II: A Retrospective.

Author information

1
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA.

Abstract

The origins of our current understanding of control of transcription elongation lie in pioneering experiments that mapped RNA polymerase II on viral and cellular genes. These studies first uncovered the surprising excess of polymerase molecules that we now know to be situated at the at the 5' ends of most genes in multicellular organisms. The pileup of pol II near transcription start sites reflects a ubiquitous bottle-neck that limits elongation right at the start of the transcription elongation. Subsequent seminal work identified conserved protein factors that positively and negatively control the flux of polymerase through this bottle-neck, and make a major contribution to control of gene expression.

14.
Mol Cell. 2012 May 11;46(3):311-24. doi: 10.1016/j.molcel.2012.03.006. Epub 2012 Apr 5.

mRNA decapping factors and the exonuclease Xrn2 function in widespread premature termination of RNA polymerase II transcription.

Author information

1
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA.

Abstract

We report a function of human mRNA decapping factors in control of transcription by RNA polymerase II. Decapping proteins Edc3, Dcp1a, and Dcp2 and the termination factor TTF2 coimmunoprecipitate with Xrn2, the nuclear 5'-3' exonuclease "torpedo" that facilitates transcription termination at the 3' ends of genes. Dcp1a, Xrn2, and TTF2 localize near transcription start sites (TSSs) by ChIP-seq. At genes with 5' peaks of paused pol II, knockdown of decapping or termination factors Xrn2 and TTF2 shifted polymerase away from the TSS toward upstream and downstream distal positions. This redistribution of pol II is similar in magnitude to that caused by depletion of the elongation factor Spt5. We propose that coupled decapping of nascent transcripts and premature termination by the "torpedo" mechanism is a widespread mechanism that limits bidirectional pol II elongation. Regulated cotranscriptional decapping near promoter-proximal pause sites followed by premature termination could control productive pol II elongation.

Comment in

PMID:
22483619
PMCID:
PMC3806456
DOI:
10.1016/j.molcel.2012.03.006
[Indexed for MEDLINE]
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15.
Nat Struct Mol Biol. 2011 Sep 25;18(10):1164-71. doi: 10.1038/nsmb.2126.

The export factor Yra1 modulates mRNA 3' end processing.

Author information

1
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado, USA.

Abstract

The Saccharomyces cerevisiae mRNA export adaptor Yra1 binds the Pcf11 subunit of cleavage-polyadenylation factor CF1A that links export to 3' end formation. We found that an unexpected consequence of this interaction is that Yra1 influences cleavage-polyadenylation. Yra1 competes with the CF1A subunit Clp1 for binding to Pcf11, and excess Yra1 inhibits 3' processing in vitro. Release of Yra1 at the 3' ends of genes coincides with recruitment of Clp1, and depletion of Yra1 enhances Clp1 recruitment within some genes. These results suggest that CF1A is not necessarily recruited as a complete unit; instead, Clp1 can be incorporated co-transcriptionally in a process regulated by Yra1. Yra1 depletion causes widespread changes in poly(A) site choice, particularly at sites where the efficiency element is divergently positioned. We propose that one way Yra1 modulates cleavage-polyadenylation is by influencing co-transcriptional assembly of the CF1A 3' processing factor.

PMID:
21947206
PMCID:
PMC3307051
DOI:
10.1038/nsmb.2126
[Indexed for MEDLINE]
Free PMC Article
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16.
Proc Natl Acad Sci U S A. 2011 Aug 16;108(33):13564-9. doi: 10.1073/pnas.1109475108. Epub 2011 Aug 1.

Pre-mRNA splicing is a determinant of histone H3K36 methylation.

Author information

1
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, University of Colorado Health Sciences Center, Aurora, CO 80045, USA.

Abstract

A chromatin code appears to mark introns and exons with distinct patterns of nucleosome enrichment and histone methylation. We investigated whether a causal relationship exists between splicing and chromatin modification by asking whether splice-site mutations affect the methylation of histone H3K36. Deletions of the 3' splice site in intron 2 or in both introns 1 and 2 of an integrated β-globin reporter gene caused a shift in relative distribution of H3K36 trimethylation away from 5' ends and toward 3' ends. The effects of splice-site mutations correlated with enhanced retention of a U5 snRNP subunit on transcription complexes downstream of the gene. In contrast, a poly(A) site mutation did not affect H3K36 methylation. Similarly, global inhibition of splicing by spliceostatin A caused a rapid repositioning of H3K36me3 away from 5' ends in favor of 3' ends. These results suggest that the cotranscriptional splicing apparatus influences establishment of normal patterns of histone modification.

PMID:
21807997
PMCID:
PMC3158196
DOI:
10.1073/pnas.1109475108
[Indexed for MEDLINE]
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17.
Nat Struct Mol Biol. 2010 Oct;17(10):1279-86. doi: 10.1038/nsmb.1913. Epub 2010 Sep 12.

Gene-specific RNA polymerase II phosphorylation and the CTD code.

Author information

1
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado, USA.

Abstract

Phosphorylation of the RNA polymerase (Pol) II C-terminal domain (CTD) repeats (1-YSPTSPS-7) is coupled to transcription and may act as a 'code' that controls mRNA synthesis and processing. To examine the code in budding yeast, we mapped genome-wide CTD Ser2, Ser5 and Ser7 phosphorylations and the CTD-associated termination factors Nrd1 and Pcf11. Phospho-CTD dynamics are not scaled to gene length and are gene-specific, with highest Ser5 and Ser7 phosphorylation at the 5' ends of well-expressed genes with nucleosome-occupied promoters. The CTD kinases Kin28 and Ctk1 markedly affect Pol II distribution in a gene-specific way. The code is therefore written differently on different genes, probably under the control of promoters. Ser7 phosphorylation is enriched on introns and at sites of Nrd1 accumulation, suggesting links to splicing and Nrd1 recruitment. Nrd1 and Pcf11 frequently colocalize, suggesting functional overlap. Unexpectedly, Pcf11 is enriched at centromeres and Pol III-transcribed genes.

PMID:
20835241
PMCID:
PMC3048030
DOI:
10.1038/nsmb.1913
[Indexed for MEDLINE]
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18.
Nat Struct Mol Biol. 2009 Sep;16(9):916-22. doi: 10.1038/nsmb.1652. Epub 2009 Aug 23.

Fast ribozyme cleavage releases transcripts from RNA polymerase II and aborts co-transcriptional pre-mRNA processing.

Author information

1
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, University of Colorado at Denver Health Sciences Center, Aurora, Colorado, USA.

Abstract

We investigated whether a continuous transcript is necessary for co-transcriptional pre-mRNA processing. Cutting an intron with the fast-cleaving hepatitis delta ribozyme, but not the slower hammerhead, inhibited splicing. Therefore, exon tethering to RNA polymerase II (Pol II) cannot rescue splicing of a transcript severed by a ribozyme that cleaves rapidly relative to the rate of splicing. Ribozyme cutting also released cap-binding complex (CBC) from the gene, suggesting that exon 1 is not tethered. Unexpectedly, cutting within exons inhibited splicing of distal introns, where exon definition is not affected, probably owing to disruption of the interactions with the CBC and the Pol II C-terminal domain that facilitate splicing. Ribozyme cutting within the mRNA also inhibited 3' processing and transcription termination. We propose that damaging the nascent transcript aborts pre-mRNA processing and that this mechanism may help to prevent association of processing factors with Pol II that is not productively engaged in transcription.

PMID:
19701200
PMCID:
PMC2755206
DOI:
10.1038/nsmb.1652
[Indexed for MEDLINE]
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19.
Mol Cell Biol. 2009 Oct;29(20):5455-64. doi: 10.1128/MCB.00637-09. Epub 2009 Aug 10.

TFIIH-associated Cdk7 kinase functions in phosphorylation of C-terminal domain Ser7 residues, promoter-proximal pausing, and termination by RNA polymerase II.

Author information

1
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, UCHSC, MS 8101, P.O. Box 6511, Aurora, CO 80045, USA.

Abstract

The function of human TFIIH-associated Cdk7 in RNA polymerase II (Pol II) transcription and C-terminal domain (CTD) phosphorylation was investigated in analogue-sensitive Cdk7(as/as) mutant cells where the kinase can be inhibited without disrupting TFIIH. We show that both Cdk7 and Cdk9/PTEFb contribute to phosphorylation of Pol II CTD Ser5 residues on transcribed genes. Cdk7 is also a major kinase of CTD Ser7 on Pol II at the c-fos and U snRNA genes. Furthermore, TFIIH and recombinant Cdk7-CycH-Mat1 as well as recombinant Cdk9-CycT1 phosphorylated CTD Ser7 and Ser5 residues in vitro. Inhibition of Cdk7 in vivo suppressed the amount of Pol II accumulated at 5' ends on several genes including c-myc, p21, and glyceraldehyde-3-phosphate dehydrogenase genes, indicating reduced promoter-proximal pausing or polymerase "leaking" into the gene. Consistent with a 5' pausing defect, Cdk7 inhibition reduced recruitment of the negative elongation factor NELF at start sites. A role of Cdk7 in regulating elongation is further suggested by enhanced histone H4 acetylation and diminished histone H4 trimethylation on lysine 36-two marks of elongation-within genes when the kinase was inhibited. Consistent with a new role for TFIIH at 3' ends, it was detected within genes and 3'-flanking regions, and Cdk7 inhibition delayed pausing and transcription termination.

PMID:
19667075
PMCID:
PMC2756882
DOI:
10.1128/MCB.00637-09
[Indexed for MEDLINE]
Free PMC Article
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20.
Mol Cell. 2009 Jan 30;33(2):215-26. doi: 10.1016/j.molcel.2008.12.007. Epub 2008 Dec 24.

Cotranscriptional recruitment of the mRNA export factor Yra1 by direct interaction with the 3' end processing factor Pcf11.

Author information

1
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA.

Abstract

We investigated recruitment of the yeast mRNA export factor Yra1 to the transcription elongation complex (TEC). Previously, the Sub2 helicase subunit of TREX was proposed to recruit Yra1. We report that Sub2 is dispensable for Yra1 recruitment, but the cleavage/polyadenylation factor, CF1A, is required. Yra1 binds directly to the Zn finger/Clp1 region of Pcf11, the pol II CTD-binding subunit of CF1A, and this interaction is conserved between their human homologs. Tethering of Pcf11 to nascent mRNA is sufficient to enhance Yra1 recruitment. Interaction with Pcf11 can therefore explain Yra1 binding to the TEC independently of Sub2. We propose that after initially binding to Pcf11, Yra1 is transferred to Sub2. Consistent with this idea, Pcf11 binds the same regions of Yra1 that also contact Sub2, indicating a mutually exclusive interaction. These results suggest a mechanism for cotranscriptional assembly of the export competent mRNP and for coordinating export with 3' end processing.

PMID:
19110458
PMCID:
PMC2659397
DOI:
10.1016/j.molcel.2008.12.007
[Indexed for MEDLINE]
Free PMC Article
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