Publications by authors named "Sylvain Egloff"

22 Publications

  • Page 1 of 1

The 7SK/P-TEFb snRNP controls ultraviolet radiation-induced transcriptional reprogramming.

Cell Rep 2021 Apr;35(2):108965

Molecular, Cellular and Developmental Biology Department (MCD), Centre de Biologie Intégrative (CBI), University of Toulouse, CNRS, UPS, 31062 Toulouse, France. Electronic address:

Conversion of promoter-proximally paused RNA polymerase II (RNAPII) into elongating polymerase by the positive transcription elongation factor b (P-TEFb) is a central regulatory step of mRNA synthesis. The activity of P-TEFb is controlled mainly by the 7SK small nuclear ribonucleoprotein (snRNP), which sequesters active P-TEFb into inactive 7SK/P-TEFb snRNP. Here we demonstrate that under normal culture conditions, the lack of 7SK snRNP has only minor impacts on global RNAPII transcription without detectable consequences on cell proliferation. However, upon ultraviolet (UV)-light-induced DNA damage, cells lacking 7SK have a defective transcriptional response and reduced viability. Both UV-induced release of "lesion-scanning" polymerases and activation of key early-responsive genes are compromised in the absence of 7SK. Proper induction of 7SK-dependent UV-responsive genes requires P-TEFb activity directly mobilized from the nucleoplasmic 7SK/P-TEFb snRNP. Our data demonstrate that the primary function of the 7SK/P-TEFb snRNP is to orchestrate the proper transcriptional response to stress.
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http://dx.doi.org/10.1016/j.celrep.2021.108965DOI Listing
April 2021

Noncoding RNAs Set the Stage for RNA Polymerase II Transcription.

Trends Genet 2021 03 9;37(3):279-291. Epub 2020 Oct 9.

Sir William Dunn School of Pathology, University of Oxford, UK. Electronic address:

Effective synthesis of mammalian messenger (m)RNAs depends on many factors that together direct RNA polymerase II (pol II) through the different stages of the transcription cycle and ensure efficient cotranscriptional processing of mRNAs. In addition to the many proteins involved in transcription initiation, elongation, and termination, several noncoding (nc)RNAs also function as global transcriptional regulators. Understanding the mode of action of these non-protein regulators has been an intense area of research in recent years. Here, we describe how these ncRNAs influence key regulatory steps of the transcription process, to affect large numbers of genes. Through direct association with pol II or by modulating the activity of transcription or RNA processing factors, these regulatory RNAs perform critical roles in gene expression.
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http://dx.doi.org/10.1016/j.tig.2020.09.013DOI Listing
March 2021

7SK small nuclear RNA, a multifunctional transcriptional regulatory RNA with gene-specific features.

Transcription 2018 4;9(2):95-101. Epub 2017 Oct 4.

a Laboratoire de Biologie Moléculaire Eucaryote du CNRS, UMR5099, Centre de Biologie Intégrative, Université Paul Sabatier , Toulouse , France.

The 7SK small nuclear RNA is a multifunctional transcriptional regulatory RNA that controls the nuclear activity of the positive transcription elongation factor b (P-TEFb), specifically targets P-TEFb to the promoter regions of selected protein-coding genes and promotes transcription of RNA polymerase II-specific spliceosomal small nuclear RNA genes.
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http://dx.doi.org/10.1080/21541264.2017.1344346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834218PMC
November 2018

The 7SK snRNP associates with the little elongation complex to promote snRNA gene expression.

EMBO J 2017 04 2;36(7):934-948. Epub 2017 Mar 2.

Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), CNRS, UPS, Université de Toulouse, Toulouse Cedex 9, France

The 7SK small nuclear RNP (snRNP), composed of the 7SK small nuclear RNA (snRNA), MePCE, and Larp7, regulates the mRNA elongation capacity of RNA polymerase II (RNAPII) through controlling the nuclear activity of positive transcription elongation factor b (P-TEFb). Here, we demonstrate that the human 7SK snRNP also functions as a canonical transcription factor that, in collaboration with the little elongation complex (LEC) comprising ELL, Ice1, Ice2, and ZC3H8, promotes transcription of RNAPII-specific spliceosomal snRNA and small nucleolar RNA (snoRNA) genes. The 7SK snRNA specifically associates with a fraction of RNAPII hyperphosphorylated at Ser5 and Ser7, which is a hallmark of RNAPII engaged in snRNA synthesis. Chromatin immunoprecipitation (ChIP) and chromatin isolation by RNA purification (ChIRP) experiments revealed enrichments for all components of the 7SK snRNP on RNAPII-specific sn/snoRNA genes. Depletion of 7SK snRNA or Larp7 disrupts LEC integrity, inhibits RNAPII recruitment to RNAPII-specific sn/snoRNA genes, and reduces nascent snRNA and snoRNA synthesis. Thus, through controlling both mRNA elongation and sn/snoRNA synthesis, the 7SK snRNP is a key regulator of nuclear RNA production by RNAPII.
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http://dx.doi.org/10.15252/embj.201695740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5376971PMC
April 2017

The pol II CTD: new twists in the tail.

Nat Struct Mol Biol 2016 09;23(9):771-7

Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

The C-terminal domain (CTD) of the large subunit of RNA polymerase (pol) II comprises conserved heptad repeats, and post-translational modification of the CTD regulates transcription and cotranscriptional RNA processing. Recently, the spatial patterns of modification of the CTD repeats have been investigated, and new functions of CTD modification have been revealed. In addition, there are new insights into the roles of the enzymes that decorate the CTD. We review these new findings and reassess the role of the pol II CTD in the regulation of gene expression.
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http://dx.doi.org/10.1038/nsmb.3285DOI Listing
September 2016

CTCF regulates NELF, DSIF and P-TEFb recruitment during transcription.

Transcription 2015 23;6(5):79-90. Epub 2015 Sep 23.

a Sir William Dunn School of Pathology; University of Oxford ; Oxford , UK.

CTCF is a versatile transcription factor with well-established roles in chromatin organization and insulator function. Recent findings also implicate CTCF in the control of elongation by RNA polymerase (RNAP) II. Here we show that CTCF knockdown abrogates RNAP II pausing at the early elongation checkpoint of c-myc by affecting recruitment of DRB-sensitivity-inducing factor (DSIF). CTCF knockdown also causes a termination defect on the U2 snRNA genes (U2), by affecting recruitment of negative elongation factor (NELF). In addition, CTCF is required for recruitment of positive elongation factor b (P-TEFb), which phosphorylates NELF, DSIF, and Ser2 of the RNAP II CTD to activate elongation of transcription of c-myc and recognition of the snRNA gene-specific 3' box RNA processing signal. These findings implicate CTCF in a complex network of protein:protein/protein:DNA interactions and assign a key role to CTCF in controlling RNAP II transcription through the elongation checkpoint of the protein-coding c-myc and the termination site of the non-coding U2, by regulating the recruitment and/or activity of key players in these processes.
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http://dx.doi.org/10.1080/21541264.2015.1095269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4802788PMC
October 2016

RNA elements directing in vivo assembly of the 7SK/MePCE/Larp7 transcriptional regulatory snRNP.

Nucleic Acids Res 2013 Apr 6;41(8):4686-98. Epub 2013 Mar 6.

Laboratoire de Biologie Moléculaire Eucaryote du CNRS, UMR5099, IFR109 CNRS, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex 9, France.

Through controlling the nuclear level of active positive transcription elongation factor b (P-TEFb), the 7SK small nuclear RNA (snRNA) functions as a key regulator of RNA polymerase II transcription. Together with hexamethylene bisacetamide-inducible proteins 1/2 (HEXIM1/2), the 7SK snRNA sequesters P-TEFb into transcriptionally inactive ribonucleoprotein (RNP). In response to transcriptional stimulation, the 7SK/HEXIM/P-TEFb RNP releases P-TEFb to promote polymerase II-mediated messenger RNA synthesis. Besides transiently associating with HEXIM1/2 and P-TEFb, the 7SK snRNA stably interacts with the La-related protein 7 (Larp7) and the methylphosphate capping enzyme (MePCE). In this study, we used in vivo RNA-protein interaction assays to determine the sequence and structural elements of human 7SK snRNA directing assembly of the 7SK/MePCE/Larp7 core snRNP. MePCE interacts with the short 5'-terminal G1-U4/U106-G111 helix-tail motif and Larp7 binds to the 3'-terminal hairpin and the following U-rich tail of 7SK. The overall RNA structure and some particular nucleotides provide the information for specific binding of MePCE and Larp7. We also demonstrate that binding of Larp7 to 7SK is a prerequisite for in vivo recruitment of P-TEFb, indicating that besides providing stability for 7SK, Larp7 directly participates in P-TEFb regulation. Our results provide further explanation for the frequently observed link between Larp7 mutations and cancer development.
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http://dx.doi.org/10.1093/nar/gkt159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632141PMC
April 2013

Role of Ser7 phosphorylation of the CTD during transcription of snRNA genes.

Authors:
Sylvain Egloff

RNA Biol 2012 Aug 1;9(8):1033-8. Epub 2012 Aug 1.

Université de Toulouse, UPS, Laboratoire de Biologie Moléculaire Eucaryote, Toulouse, France.

The largest subunit of RNA polymerase (pol) II, Rpb1, contains an unusual carboxyl-terminal domain (CTD) composed of consecutive repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser (Y 1S 2P 3T 4S 5P 6S 7). During transcription, Ser2, Ser5 and Ser7 are subjected to dynamic phosphorylation and dephosphorylation by CTD kinases and phosphatases, creating a characteristic CTD phosphorylation pattern along genes. This CTD "code" allows the coupling of transcription with co-transcriptional RNA processing, through the timely recruitment of the appropriate factors at the right point of the transcription cycle. In mammals, phosphorylation of Ser7 (Ser7P) is detected on all pol II-transcribed genes, but is only essential for expression of a sub-class of genes encoding small nuclear (sn)RNAs. The molecular mechanisms by which Ser7P influences expression of these particular genes are becoming clearer. Here, I discuss our recent findings clarifying how Ser7P facilitates transcription of these genes and 3'end processing of the transcripts, through recruitment of the RPAP2 phosphatase and the snRNA gene-specific Integrator complex.
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http://dx.doi.org/10.4161/rna.21166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3551856PMC
August 2012

Updating the RNA polymerase CTD code: adding gene-specific layers.

Trends Genet 2012 Jul 21;28(7):333-41. Epub 2012 May 21.

Université de Toulouse, UPS, Laboratoire de Biologie Moléculaire Eucaryote, F-31000 Toulouse, France.

The carboxyl-terminal domain (CTD) of RNA polymerase (pol) II comprises multiple tandem repeats with the consensus sequence Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7) that can be extensively and reversibly modified in vivo. CTD modifications orchestrate the interplay between transcription and processing of mRNA. Although phosphorylation of Ser2 (Ser2P) and Ser5 (Ser5P) residues has been described as being essential for the expression of most pol II-transcribed genes, recent findings highlight gene-specific effects of newly discovered CTD modifications. Here, we incorporate these latest findings in an updated review of the currently known elements that contribute to the CTD code and how it is recognized by proteins involved in transcription and RNA maturation. As modification of the CTD has a major impact on gene expression, a better understanding of the CTD code is integral to the understanding of how gene expression is regulated.
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http://dx.doi.org/10.1016/j.tig.2012.03.007DOI Listing
July 2012

[Misregulation of P-TEFb activity: pathological consequences].

Med Sci (Paris) 2012 Feb 27;28(2):200-5. Epub 2012 Feb 27.

Université de Toulouse, université Paul Sabatier, CNRS laboratoire de biologie moléculaire des eucaryotes, Toulouse, France.

P-TEFb stimulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA pol II and antagonizing the effects of negative elongation factors. Its cellular availability is controlled by an abundant non coding RNA, conserved through evolution, the 7SK RNA. Together with the HEXIM proteins, 7SK RNA associates with and sequesters a fraction of cellular P-TEFb into a catalytically inactive complex. Active and inactive forms of P-TEFb are kept in a functional and dynamic equilibrium tightly linked to the transcriptional requirement of the cell. Importantly, cardiac hypertrophy and development of various types of human malignancies have been associated with increased P-TEFb activity, consequence of a disruption of this regulatory equilibrium. In addition, the HIV-1 Tat protein also releases P-TEFb from the 7SK/HEXIM complex during viral infection to promote viral transcription and replication. Here, we review the roles played by the 7SK RNP in cancer development, cardiac hypertrophy and AIDS.
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http://dx.doi.org/10.1051/medsci/2012282019DOI Listing
February 2012

Ser7 phosphorylation of the CTD recruits the RPAP2 Ser5 phosphatase to snRNA genes.

Mol Cell 2012 Jan 1;45(1):111-22. Epub 2011 Dec 1.

Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK.

The carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II) comprises multiple heptapeptide repeats of the consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Reversible phosphorylation of Ser2, Ser5, and Ser7 during the transcription cycle mediates the sequential recruitment of transcription/RNA processing factors. Phosphorylation of Ser7 is required for recruitment of the gene type-specific Integrator complex to the Pol II-transcribed small nuclear (sn)RNA genes. Here, we show that RNA Pol II-associated protein 2 (RPAP2) specifically recognizes the phospho-Ser7 mark on the Pol II CTD and also interacts with Integrator subunits. siRNA-mediated knockdown of RPAP2 and mutation of Ser7 to alanine cause similar defects in snRNA gene expression. In addition, we show that RPAP2 is a CTD Ser5 phosphatase. Taken together, our results indicate that during transcription of snRNA genes, Ser7 phosphorylation facilitates recruitment of RPAP2, which in turn both recruits Integrator and dephosphorylates Ser5.
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http://dx.doi.org/10.1016/j.molcel.2011.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3262128PMC
January 2012

Controlling cellular P-TEFb activity by the HIV-1 transcriptional transactivator Tat.

PLoS Pathog 2010 Oct 14;6(10):e1001152. Epub 2010 Oct 14.

Laboratoire de Biologie Moléculaire Eucaryote du CNRS, UMR5099, IFR109 CNRS, Université Paul Sabatier, Toulouse, France.

The human immunodeficiency virus 1 (HIV-1) transcriptional transactivator (Tat) is essential for synthesis of full-length transcripts from the integrated viral genome by RNA polymerase II (Pol II). Tat recruits the host positive transcription elongation factor b (P-TEFb) to the HIV-1 promoter through binding to the transactivator RNA (TAR) at the 5'-end of the nascent HIV transcript. P-TEFb is a general Pol II transcription factor; its cellular activity is controlled by the 7SK small nuclear RNA (snRNA) and the HEXIM1 protein, which sequester P-TEFb into transcriptionally inactive 7SK/HEXIM/P-TEFb snRNP. Besides targeting P-TEFb to HIV transcription, Tat also increases the nuclear level of active P-TEFb through promoting its dissociation from the 7SK/HEXIM/P-TEFb RNP by an unclear mechanism. In this study, by using in vitro and in vivo RNA-protein binding assays, we demonstrate that HIV-1 Tat binds with high specificity and efficiency to an evolutionarily highly conserved stem-bulge-stem motif of the 5'-hairpin of human 7SK snRNA. The newly discovered Tat-binding motif of 7SK is structurally and functionally indistinguishable from the extensively characterized Tat-binding site of HIV TAR and importantly, it is imbedded in the HEXIM-binding elements of 7SK snRNA. We show that Tat efficiently replaces HEXIM1 on the 7SK snRNA in vivo and therefore, it promotes the disassembly of the 7SK/HEXIM/P-TEFb negative transcriptional regulatory snRNP to augment the nuclear level of active P-TEFb. This is the first demonstration that HIV-1 specifically targets an important cellular regulatory RNA, most probably to promote viral transcription and replication. Demonstration that the human 7SK snRNA carries a TAR RNA-like Tat-binding element that is essential for the normal transcriptional regulatory function of 7SK questions the viability of HIV therapeutic approaches based on small drugs blocking the Tat-binding site of HIV TAR.
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http://dx.doi.org/10.1371/journal.ppat.1001152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2954905PMC
October 2010

The integrator complex recognizes a new double mark on the RNA polymerase II carboxyl-terminal domain.

J Biol Chem 2010 Jul 10;285(27):20564-9. Epub 2010 May 10.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) comprises multiple tandem repeats of the heptapeptide Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7). This unusual structure serves as a platform for the binding of factors required for expression of pol II-transcribed genes, including the small nuclear RNA (snRNA) gene-specific Integrator complex. The pol II CTD specifically mediates recruitment of Integrator to the promoter of snRNA genes to activate transcription and direct 3' end processing of the transcripts. Phosphorylation of the CTD and a serine in position 7 are necessary for Integrator recruitment. Here, we have further investigated the requirement of the serines in the CTD heptapeptide and their phosphorylation for Integrator binding. We show that both Ser(2) and Ser(7) of the CTD are required and that phosphorylation of these residues is necessary and sufficient for efficient binding. Using synthetic phosphopeptides, we have determined the pattern of the minimal Ser(2)/Ser(7) double phosphorylation mark required for Integrator to interact with the CTD. This novel double phosphorylation mark is a new addition to the functional repertoire of the CTD code and may be a specific signal for snRNA gene expression.
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http://dx.doi.org/10.1074/jbc.M110.132530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2898319PMC
July 2010

Chromatin structure is implicated in "late" elongation checkpoints on the U2 snRNA and beta-actin genes.

Mol Cell Biol 2009 Jul 18;29(14):4002-13. Epub 2009 May 18.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom.

The negative elongation factor NELF is a key component of an early elongation checkpoint generally located within 100 bp of the transcription start site of protein-coding genes. Negotiation of this checkpoint and conversion to productive elongation require phosphorylation of the carboxy-terminal domain of RNA polymerase II (pol II), NELF, and DRB sensitivity-inducing factor (DSIF) by positive transcription elongation factor b (P-TEFb). P-TEFb is dispensable for transcription of the noncoding U2 snRNA genes, suggesting that a NELF-dependent checkpoint is absent. However, we find that NELF at the end of the 800-bp U2 gene transcription unit and RNA interference-mediated knockdown of NELF causes a termination defect. NELF is also associated 800 bp downstream of the transcription start site of the beta-actin gene, where a "late" P-TEFb-dependent checkpoint occurs. Interestingly, both genes have an extended nucleosome-depleted region up to the NELF-dependent control point. In both cases, transcription through this region is P-TEFb independent, implicating chromatin in the formation of the terminator/checkpoint. Furthermore, CTCF colocalizes with NELF on the U2 and beta-actin genes, raising the possibility that it helps the positioning and/or function of the NELF-dependent control point on these genes.
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http://dx.doi.org/10.1128/MCB.00189-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2704739PMC
July 2009

Expression of human snRNA genes from beginning to end.

Biochem Soc Trans 2008 Aug;36(Pt 4):590-4

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, UK.

In addition to protein-coding genes, mammalian pol II (RNA polymerase II) transcribes independent genes for some non-coding RNAs, including the spliceosomal U1 and U2 snRNAs (small nuclear RNAs). snRNA genes differ from protein-coding genes in several key respects and some of the mechanisms involved in expression are gene-type-specific. For example, snRNA gene promoters contain an essential PSE (proximal sequence element) unique to these genes, the RNA-encoding regions contain no introns, elongation of transcription is P-TEFb (positive transcription elongation factor b)-independent and RNA 3'-end formation is directed by a 3'-box rather than a cleavage and polyadenylation signal. However, the CTD (C-terminal domain) of pol II closely couples transcription with RNA 5' and 3' processing in expression of both gene types. Recently, it was shown that snRNA promoter-specific recognition of the 3'-box RNA processing signal requires a novel phosphorylation mark on the pol II CTD. This new mark plays a critical role in the recruitment of the snRNA gene-specific RNA-processing complex, Integrator. These new findings provide the first example of a phosphorylation mark on the CTD heptapeptide that can be read in a gene-type-specific manner, reinforcing the notion of a CTD code. Here, we review the control of expression of snRNA genes from initiation to termination of transcription.
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http://dx.doi.org/10.1042/BST0360590DOI Listing
August 2008

Role of the C-terminal domain of RNA polymerase II in expression of small nuclear RNA genes.

Biochem Soc Trans 2008 Jun;36(Pt 3):537-9

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

Pol II (RNA polymerase II) transcribes the genes encoding proteins and non-coding snRNAs (small nuclear RNAs). The largest subunit of Pol II contains a distinctive CTD (C-terminal domain) comprising a repetitive heptad amino acid sequence, Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7). This domain is now known to play a major role in the processes of transcription and co-transcriptional RNA processing in expression of both snRNA and protein-coding genes. The heptapeptide repeat unit can be extensively modified in vivo and covalent modifications of the CTD during the transcription cycle result in the ordered recruitment of RNA-processing factors. The most studied modifications are the phosphorylation of the serine residues in position 2 and 5 (Ser(2) and Ser(5)), which play an important role in the co-transcriptional processing of both mRNA and snRNA. An additional, recently identified CTD modification, phosphorylation of the serine residue in position 7 (Ser(7)) of the heptapeptide, is however specifically required for expression of snRNA genes. These findings provide interesting insights into the control of gene-specific Pol II function.
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http://dx.doi.org/10.1042/BST0360537DOI Listing
June 2008

Cracking the RNA polymerase II CTD code.

Trends Genet 2008 Jun 3;24(6):280-8. Epub 2008 May 3.

Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II comprises multiple tandem conserved heptapeptide repeats, unique to this eukaryotic RNA polymerase. This unusual structure provides a docking platform for factors involved in various co-transcriptional events. Recruitment of the appropriate factors at different stages of the transcription cycle is achieved through changing patterns of post-translational modification of the CTD repeats, which create a readable 'code'. A new phosphorylation mark both expands the CTD code and provides the first example of a CTD signal read in a gene type-specific manner. How and when is the code written and read? How does it contribute to transcription and coordinate RNA processing?
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http://dx.doi.org/10.1016/j.tig.2008.03.008DOI Listing
June 2008

Serine-7 of the RNA polymerase II CTD is specifically required for snRNA gene expression.

Science 2007 Dec;318(5857):1777-9

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

RNA polymerase II (Pol II) transcribes genes that encode proteins and noncoding small nuclear RNAs (snRNAs). The carboxyl-terminal repeat domain (CTD) of the largest subunit of mammalian RNA Pol II, comprising tandem repeats of the heptapeptide consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, is required for expression of both gene types. We show that mutation of serine-7 to alanine causes a specific defect in snRNA gene expression. We also present evidence that phosphorylation of serine-7 facilitates interaction with the snRNA gene-specific Integrator complex. These findings assign a biological function to this amino acid and highlight a gene type-specific requirement for a residue within the CTD heptapeptide, supporting the existence of a CTD code.
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http://dx.doi.org/10.1126/science.1145989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2263945PMC
December 2007

Dynamic remodelling of human 7SK snRNP controls the nuclear level of active P-TEFb.

EMBO J 2007 Aug 5;26(15):3570-80. Epub 2007 Jul 5.

Laboratoire de Biologie Moléculaire Eucaryote, UMR5099, CNRS-Université Paul Sabatier, Toulouse, France.

The 7SK small nuclear RNA (snRNA) regulates RNA polymerase II transcription elongation by controlling the protein kinase activity of the positive transcription elongation factor b (P-TEFb). In cooperation with HEXIM1, the 7SK snRNA sequesters P-TEFb into the kinase-inactive 7SK/HEXIM1/P-TEFb small nuclear ribonucleoprotein (snRNP), and thereby, controls the nuclear level of active P-TEFb. Here, we report that a fraction of HeLa 7SK snRNA that is not involved in 7SK/HEXIM1/P-TEFb formation, specifically interacts with RNA helicase A (RHA), heterogeneous nuclear ribonucleoprotein A1 (hnRNP), A2/B1, R and Q proteins. Inhibition of cellular transcription induces disassembly of 7SK/HEXIM1/P-TEFb and at the same time, increases the level of 7SK snRNPs containing RHA, hnRNP A1, A2/B1, R and Q. Removal of transcription inhibitors restores the original levels of the 7SK/HEXIM1/P-TEFb and '7SK/hnRNP' complexes. 7SK/HEXIM1/P-TEFb snRNPs containing mutant 7SK RNAs lacking the capacity for binding hnRNP A1, A2, R and Q are resistant to stress-induced disassembly, indicating that recruitment of the novel 7SK snRNP proteins is essential for disruption of 7SK/HEXIM1/P-TEFb. Thus, we propose that the nuclear level of active P-TEFb is controlled by dynamic and reversible remodelling of 7SK snRNP.
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http://dx.doi.org/10.1038/sj.emboj.7601783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1949012PMC
August 2007

Regulation of polymerase II transcription by 7SK snRNA: two distinct RNA elements direct P-TEFb and HEXIM1 binding.

Mol Cell Biol 2006 Jan;26(2):630-42

Laboratoire de Biologie Moléculaire Eucaryote du CNRS, UMR5099 and Université Paul Sabatier, IFR109, 118 route de Narbonne, 31062 Toulouse Cedex 4, France.

The positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1/T2, stimulates transcription by phosphorylating RNA polymerase II. The 7SK small nuclear RNA, in cooperation with HEXIM1 protein, functions as a general polymerase II transcription regulator by sequestering P-TEFb into a large kinase-inactive 7SK/HEXIM1/P-TEFb complex. Here, determination and characterization of the functionally essential elements of human 7SK snRNA directing HEXIM1 and P-TEFb binding led to a new model for the assembly of the 7SK/HEXIM1/P-TEFb regulatory complex. We demonstrate that two structurally and functionally distinct protein binding elements located in the 5'- and 3'-terminal hairpins of 7SK support the in vivo recruitment of HEXIM1 and P-TEFb. Consistently, a minimal regulatory RNA composed of the 5' and 3' hairpins of 7SK can modulate polymerase II transcription in HeLa cells. HEXIM1 binds independently and specifically to the G24-C48/G60-C87 distal segment of the 5' hairpin of 7SK. Binding of HEXIM1 is a prerequisite for association of P-TEFb with the G302-C324 apical region of the 3' hairpin of 7SK that is highly reminiscent of the human immunodeficiency virus transactivation-responsive RNA.
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http://dx.doi.org/10.1128/MCB.26.2.630-642.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1346915PMC
January 2006

Probing plasmid partition with centromere-based incompatibility.

Mol Microbiol 2005 Jan;55(2):511-25

Laboratoire de Microbiologie et Génétique Moléculaire, CNRS, 118 route de Narbonne, 31062 Toulouse, France.

Low-copy number plasmids of bacteria rely on specific centromeres for regular partition into daughter cells. When also present on a second plasmid, the centromere can render the two plasmids incompatible, disrupting partition and causing plasmid loss. We have investigated the basis of incompatibility exerted by the F plasmid centromere, sopC, to probe the mechanism of partition. Measurements of the effects of sopC at various gene dosages on destabilization of mini-F, on repression of the sopAB operon and on occupancy of mini-F DNA by the centromere-binding protein, SopB, revealed that among mechanisms previously proposed, no single one fully explained incompatibility. sopC on multicopy plasmids depleted SopB by titration and by contributing to repression. The resulting SopB deficit is proposed to delay partition complex formation and facilitate pairing between mini-F and the centromere vector, thereby increasing randomization of segregation. Unexpectedly, sopC on mini-P1 exerted strong incompatibility if the P1 parABS locus was absent. A mutation preventing the P1 replication initiation protein from pairing (handcuffing) reduced this strong incompatibility to the level expected for random segregation. The results indicate the importance of kinetic considerations and suggest that mini-F handcuffing promotes pairing of SopB-sopC complexes that can subsequently segregate as intact aggregates.
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http://dx.doi.org/10.1111/j.1365-2958.2004.04396.xDOI Listing
January 2005

Cooperative dimerization of the POU domain protein Brn-2 on a new motif activates the neuronal promoter of the human aromatic L-amino acid decarboxylase gene.

Brain Res Mol Brain Res 2004 Jan;120(2):151-63

Laboratoire de Biologie Moléculaire Eucaryote, CNRS UMR 5099/IFR 109, 118 route de Narbonne, 31062, Cedex, Toulouse, France.

The neuronal promoter of the human aromatic L-amino acid decarboxylase (AADC) gene contains a perfectly palindromic element (TB) that conforms to the structure of a POU domain protein binding site of the MORE+2 type. The TB motif (located at nts -900/-872 relative to the neuronal cap site) bears striking similarities with the dimeric Pit-1 binding site from growth hormone gene promoter (GH-1), and it enhanced the activity of the minimal tk promoter in transfected SK-N-BE neuroblastoma cells. In transfected COS-7 cells, the expression of a 3xTB-tk-luc was stimulated up to 11-fold by the overexpressed Brn-2 protein. In AADC gene neuronal promoter, we previously characterized a bipartite regulatory element (ONF for octamer-like/NF-Y, nts -86/-57) that binds Brn-2 and NF-Y proteins in a cooperative manner. We now show that both TB and ONF sites participate in the activation of the neuronal promoter by Brn-2. EMSA experiments showed that the recombinant Brn-2 POU domain dimerized on the TB element in a cooperative manner. By site directed mutagenesis of the POU domain of Brn-2, the dimerization interface on the TB element was localized to the hydrophobic pocket of the POU specific domain and the C-terminal part of the POU homeodomain.
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http://dx.doi.org/10.1016/j.molbrainres.2003.10.016DOI Listing
January 2004