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Olivier Bensaude - One of the best experts on this subject based on the ideXlab platform.
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an RNAi screen for aire cofactors reveals a role for hnrnpl in polymerase release and aire activated ectopic transcription
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Olivier Bensaude, Matthieu Giraud, Nada Jmari, Lina Du, Floriane Carallis, Thomas J F Nieland, Flor M Perezcampo, David E Root, Nir HacohenAbstract:Aire induces the expression of a large set of autoantigen genes in the thymus, driving immunological tolerance in maturing T cells. To determine the full spectrum of molecular mechanisms underlying the Aire transactivation function, we screened an AIRE-dependent gene-expression system with a genome-scale lentiviral shRNA library, targeting factors associated with chromatin architecture/function, transcription, and mRNA processing. Fifty-one functional allies were identified, with a preponderance of factors that impact transcriptional elongation compared with initiation, in particular members of the positive transcription elongation factor b (P-TEFb) involved in the release of “paused” RNA polymerases (CCNT2 and HEXIM1); mRNA processing and polyadenylation factors were also highlighted (HNRNPL/F, SFRS1, SFRS3, and CLP1). Aire’s functional allies were validated on transfected and endogenous target genes, including the generation of lentigenic knockdown (KD) mice. We uncovered the effect of the splicing factor Hnrnpl on Aire-induced transcription. Transcripts sensitive to the P-TEFb inhibitor flavopiridol were reduced by Hnrnpl knockdown in thymic epithelial cells, independently of their dependence on Aire, therefore indicating a general effect of Hnrnpl on RNA elongation. This conclusion was substantiated by demonstration of HNRNPL interactions with P-TEFb components (CDK9, CCNT2, HEXIM1, and the small 7SK RNA). Aire-containing complexes include 7SK RNA, the latter interaction disrupted by HNRNPL knockdown, suggesting that HNRNPL may partake in delivering inactive P-TEFb to Aire. Thus, these results indicate that mRNA processing factors cooperate with Aire to release stalled polymerases and to activate ectopic expression of autoantigen genes in the thymus.
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transcription dependent association of multiple positive transcription elongation factor units to a hexim multimer
Journal of Biological Chemistry, 2005Co-Authors: Cyprien Dulac, Van Trung Nguyen, Annemieke A Michels, Luigi Lania, Alessandro Fraldi, Francois Bonnet, Giuliana Napolitano, Olivier BensaudeAbstract:The positive transcription elongation factor (P-TEFb) comprises a kinase, CDK9, and a Cyclin T1 or T2. Its activity is inhibited by association with the HEXIM1 or HEXIM2 protein bound to 7SK small nuclear RNA. HEXIM1 and HEXIM2 were found to form stable homo- and hetero-oligomers. Using yeast two-hybrid and transfection assays, we have now shown that the C-terminal domains of HEXIM proteins directly interact with each other. Hydrodynamic parameters measured by glycerol gradient ultracentrifugation and gel-permeation chromatography demonstrate that both purified recombinant and cellular HEXIM1 proteins form highly anisotropic particles. Chemical cross-links suggest that HEXIM1 proteins form dimers. The multimeric nature of HEXIM1 is maintained in P-TEFb·HEXIM1·7SK RNA complexes. Multiple P-TEFb modules are found in the inactive P-TEFb·HEXIM1·7SK complexes. It is proposed that 7SK RNA binding to a HEXIM1 multimer promotes the simultaneous recruitment and hence inactivation of multiple P-TEFb units.
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maq1 and 7SK RNA interact with cdk9 cyclin t complexes in a transcription dependent manner
Molecular and Cellular Biology, 2003Co-Authors: Annemieke A Michels, Van Trung Nguyen, Luigi Lania, Alessandro Fraldi, Francois Bonnet, Valerie Labas, Mia Edwards, Olivier BensaudeAbstract:Phosphorylation of the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) is a critical step required for transcription elongation (7) and for recruitment of the machinery involved in pre-mRNA maturation (3, 26, 46). The CTD is unphosphorylated when RNAP II assembles onto promoters (RNAP IIA). A class of negative transcription factors including the 5,6-dichlorozo-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor and the negative elongation factor causes transcriptional arrest shortly after initiation, during which the polymerase may fall off (60). To release this block, the CTD must be phosphorylated (RNAP IIO) by positive transcription elongation factor b (P-TEFb), a protein complex that comprises cyclin-dependent kinase 9 (CDK9) and a cyclin (T1 or T2) (45). P-TEFb kinase activity is required for transcription of most class II genes (6). The human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter uses a unique mechanism: the level of proviral DNA transcription is determined by recruitment of P-TEFb to the TAR (transactivation response) element, an RNA stem-loop structure that forms at the 5′ end of the viral transcript (4, 38, 59, 66). The viral genome encodes a very potent transactivator of its own transcription, the Tat protein. The formation of a quateRNAry complex among CDK9, cyclin T1, Tat, and TAR RNA determines the recruitment of human P-TEFb to the transcription elongation complex and the efficient synthesis of long productive viral transcripts (15, 18, 30, 33, 44, 65). Binding of the 7SK small nuclear RNA (snRNA) to P-TEFb has recently been shown to be associated with the inhibition of CDK9 kinase activity (41, 62). Core P-TEFb is active, whereas the P-TEFb/7SK RNA complex is inactive. P-TEFb and 7SK associate in a reversible manner. Inhibition of cellular transcription by chemical agents or UV irradiation triggers the complete disruption of the P-TEFb/7SK complex and enhances CDK9 activity. In this study, we searched for additional cellular proteins that may be present in the P-TEFb/7SK RNA complex. A single novel P-TEFb subunit was found and termed MAQ1 (for menage a quatre), alluding to MAT1 (for menage a trois), which associates with CDK9-related CDK7 and cyclin H (10). The transcription-dependent interaction of P-TEFb with 7SK and MAQ1 may contribute to a feedback loop that modulates the activity of RNAP II.
Annemieke A Michels - One of the best experts on this subject based on the ideXlab platform.
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transcription dependent association of multiple positive transcription elongation factor units to a hexim multimer
Journal of Biological Chemistry, 2005Co-Authors: Cyprien Dulac, Van Trung Nguyen, Annemieke A Michels, Luigi Lania, Alessandro Fraldi, Francois Bonnet, Giuliana Napolitano, Olivier BensaudeAbstract:The positive transcription elongation factor (P-TEFb) comprises a kinase, CDK9, and a Cyclin T1 or T2. Its activity is inhibited by association with the HEXIM1 or HEXIM2 protein bound to 7SK small nuclear RNA. HEXIM1 and HEXIM2 were found to form stable homo- and hetero-oligomers. Using yeast two-hybrid and transfection assays, we have now shown that the C-terminal domains of HEXIM proteins directly interact with each other. Hydrodynamic parameters measured by glycerol gradient ultracentrifugation and gel-permeation chromatography demonstrate that both purified recombinant and cellular HEXIM1 proteins form highly anisotropic particles. Chemical cross-links suggest that HEXIM1 proteins form dimers. The multimeric nature of HEXIM1 is maintained in P-TEFb·HEXIM1·7SK RNA complexes. Multiple P-TEFb modules are found in the inactive P-TEFb·HEXIM1·7SK complexes. It is proposed that 7SK RNA binding to a HEXIM1 multimer promotes the simultaneous recruitment and hence inactivation of multiple P-TEFb units.
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maq1 and 7SK RNA interact with cdk9 cyclin t complexes in a transcription dependent manner
Molecular and Cellular Biology, 2003Co-Authors: Annemieke A Michels, Van Trung Nguyen, Luigi Lania, Alessandro Fraldi, Francois Bonnet, Valerie Labas, Mia Edwards, Olivier BensaudeAbstract:Phosphorylation of the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) is a critical step required for transcription elongation (7) and for recruitment of the machinery involved in pre-mRNA maturation (3, 26, 46). The CTD is unphosphorylated when RNAP II assembles onto promoters (RNAP IIA). A class of negative transcription factors including the 5,6-dichlorozo-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor and the negative elongation factor causes transcriptional arrest shortly after initiation, during which the polymerase may fall off (60). To release this block, the CTD must be phosphorylated (RNAP IIO) by positive transcription elongation factor b (P-TEFb), a protein complex that comprises cyclin-dependent kinase 9 (CDK9) and a cyclin (T1 or T2) (45). P-TEFb kinase activity is required for transcription of most class II genes (6). The human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter uses a unique mechanism: the level of proviral DNA transcription is determined by recruitment of P-TEFb to the TAR (transactivation response) element, an RNA stem-loop structure that forms at the 5′ end of the viral transcript (4, 38, 59, 66). The viral genome encodes a very potent transactivator of its own transcription, the Tat protein. The formation of a quateRNAry complex among CDK9, cyclin T1, Tat, and TAR RNA determines the recruitment of human P-TEFb to the transcription elongation complex and the efficient synthesis of long productive viral transcripts (15, 18, 30, 33, 44, 65). Binding of the 7SK small nuclear RNA (snRNA) to P-TEFb has recently been shown to be associated with the inhibition of CDK9 kinase activity (41, 62). Core P-TEFb is active, whereas the P-TEFb/7SK RNA complex is inactive. P-TEFb and 7SK associate in a reversible manner. Inhibition of cellular transcription by chemical agents or UV irradiation triggers the complete disruption of the P-TEFb/7SK complex and enhances CDK9 activity. In this study, we searched for additional cellular proteins that may be present in the P-TEFb/7SK RNA complex. A single novel P-TEFb subunit was found and termed MAQ1 (for menage a quatre), alluding to MAT1 (for menage a trois), which associates with CDK9-related CDK7 and cyclin H (10). The transcription-dependent interaction of P-TEFb with 7SK and MAQ1 may contribute to a feedback loop that modulates the activity of RNAP II.
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7SK small nuclear RNA binds to and inhibits the activity of cdk9 cyclin t complexes
Nature, 2001Co-Authors: Van Trung Nguyen, Tamás Kiss, Annemieke A MichelsAbstract:The transcription of eukaryotic protein-coding genes involves complex regulation of RNA polymerase (Pol) II activity in response to physiological conditions and developmental cues. One element of this regulation involves phosphorylation of the carboxy-terminal domain (CTD) of the largest polymerase subunit by a transcription elongation factor, P-TEFb, which comprises the kinase CDK9 and cyclin T1 or T2 (ref. 1). Here we report that in human HeLa cells more than half of the P-TEFb is sequestered in larger complexes that also contain 7SK RNA, an abundant, small nuclear RNA (snRNA) of hitherto unknown function2,3. P-TEFb and 7SK associate in a specific and reversible manner. In contrast to the smaller P-TEFb complexes, which have a high kinase activity, the larger 7SK/P-TEFb complexes show very weak kinase activity. Inhibition of cellular transcription by chemical agents or ultraviolet irradiation trigger the complete disruption of the P-TEFb/7SK complex, and enhance CDK9 activity. The transcription-dependent interaction of P-TEFb with 7SK may therefore contribute to an important feedback loop modulating the activity of RNA Pol II.
Van Trung Nguyen - One of the best experts on this subject based on the ideXlab platform.
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transcription dependent association of multiple positive transcription elongation factor units to a hexim multimer
Journal of Biological Chemistry, 2005Co-Authors: Cyprien Dulac, Van Trung Nguyen, Annemieke A Michels, Luigi Lania, Alessandro Fraldi, Francois Bonnet, Giuliana Napolitano, Olivier BensaudeAbstract:The positive transcription elongation factor (P-TEFb) comprises a kinase, CDK9, and a Cyclin T1 or T2. Its activity is inhibited by association with the HEXIM1 or HEXIM2 protein bound to 7SK small nuclear RNA. HEXIM1 and HEXIM2 were found to form stable homo- and hetero-oligomers. Using yeast two-hybrid and transfection assays, we have now shown that the C-terminal domains of HEXIM proteins directly interact with each other. Hydrodynamic parameters measured by glycerol gradient ultracentrifugation and gel-permeation chromatography demonstrate that both purified recombinant and cellular HEXIM1 proteins form highly anisotropic particles. Chemical cross-links suggest that HEXIM1 proteins form dimers. The multimeric nature of HEXIM1 is maintained in P-TEFb·HEXIM1·7SK RNA complexes. Multiple P-TEFb modules are found in the inactive P-TEFb·HEXIM1·7SK complexes. It is proposed that 7SK RNA binding to a HEXIM1 multimer promotes the simultaneous recruitment and hence inactivation of multiple P-TEFb units.
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maq1 and 7SK RNA interact with cdk9 cyclin t complexes in a transcription dependent manner
Molecular and Cellular Biology, 2003Co-Authors: Annemieke A Michels, Van Trung Nguyen, Luigi Lania, Alessandro Fraldi, Francois Bonnet, Valerie Labas, Mia Edwards, Olivier BensaudeAbstract:Phosphorylation of the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) is a critical step required for transcription elongation (7) and for recruitment of the machinery involved in pre-mRNA maturation (3, 26, 46). The CTD is unphosphorylated when RNAP II assembles onto promoters (RNAP IIA). A class of negative transcription factors including the 5,6-dichlorozo-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor and the negative elongation factor causes transcriptional arrest shortly after initiation, during which the polymerase may fall off (60). To release this block, the CTD must be phosphorylated (RNAP IIO) by positive transcription elongation factor b (P-TEFb), a protein complex that comprises cyclin-dependent kinase 9 (CDK9) and a cyclin (T1 or T2) (45). P-TEFb kinase activity is required for transcription of most class II genes (6). The human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter uses a unique mechanism: the level of proviral DNA transcription is determined by recruitment of P-TEFb to the TAR (transactivation response) element, an RNA stem-loop structure that forms at the 5′ end of the viral transcript (4, 38, 59, 66). The viral genome encodes a very potent transactivator of its own transcription, the Tat protein. The formation of a quateRNAry complex among CDK9, cyclin T1, Tat, and TAR RNA determines the recruitment of human P-TEFb to the transcription elongation complex and the efficient synthesis of long productive viral transcripts (15, 18, 30, 33, 44, 65). Binding of the 7SK small nuclear RNA (snRNA) to P-TEFb has recently been shown to be associated with the inhibition of CDK9 kinase activity (41, 62). Core P-TEFb is active, whereas the P-TEFb/7SK RNA complex is inactive. P-TEFb and 7SK associate in a reversible manner. Inhibition of cellular transcription by chemical agents or UV irradiation triggers the complete disruption of the P-TEFb/7SK complex and enhances CDK9 activity. In this study, we searched for additional cellular proteins that may be present in the P-TEFb/7SK RNA complex. A single novel P-TEFb subunit was found and termed MAQ1 (for menage a quatre), alluding to MAT1 (for menage a trois), which associates with CDK9-related CDK7 and cyclin H (10). The transcription-dependent interaction of P-TEFb with 7SK and MAQ1 may contribute to a feedback loop that modulates the activity of RNAP II.
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7SK small nuclear RNA binds to and inhibits the activity of cdk9 cyclin t complexes
Nature, 2001Co-Authors: Van Trung Nguyen, Tamás Kiss, Annemieke A MichelsAbstract:The transcription of eukaryotic protein-coding genes involves complex regulation of RNA polymerase (Pol) II activity in response to physiological conditions and developmental cues. One element of this regulation involves phosphorylation of the carboxy-terminal domain (CTD) of the largest polymerase subunit by a transcription elongation factor, P-TEFb, which comprises the kinase CDK9 and cyclin T1 or T2 (ref. 1). Here we report that in human HeLa cells more than half of the P-TEFb is sequestered in larger complexes that also contain 7SK RNA, an abundant, small nuclear RNA (snRNA) of hitherto unknown function2,3. P-TEFb and 7SK associate in a specific and reversible manner. In contrast to the smaller P-TEFb complexes, which have a high kinase activity, the larger 7SK/P-TEFb complexes show very weak kinase activity. Inhibition of cellular transcription by chemical agents or ultraviolet irradiation trigger the complete disruption of the P-TEFb/7SK complex, and enhance CDK9 activity. The transcription-dependent interaction of P-TEFb with 7SK may therefore contribute to an important feedback loop modulating the activity of RNA Pol II.
Peter F. Stadler - One of the best experts on this subject based on the ideXlab platform.
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Detailed secondary structure models of invertebrate 7SK RNAs
RNA Biology, 2017Co-Authors: Ali M. Yazbeck, Kifah Tout, Peter F. StadlerAbstract:ABSTRACTThe 7SK RNA is a small nuclear RNA that is involved in the regulation of Pol-II transcription. It is very well conserved in vertebrates, but shows extensive variations in both sequence and structure across invertebrates. A systematic homology search extended the collection of 7SK genes in both Arthropods and Lophotrochozoa making use of the large number of recently published invertebrate genomes. The extended data set made it possible to infer complete consensus structures for invertebrate 7SK RNAs. These show that not only the well-conserved 5′- and 3′- domains but all the interior Stem A domain is universally conserved. In contrast, Stem B region exhibits substantial structural variation and does not adhere to a common structural model beyond phylum level.
Alessandro Fraldi - One of the best experts on this subject based on the ideXlab platform.
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transcription dependent association of multiple positive transcription elongation factor units to a hexim multimer
Journal of Biological Chemistry, 2005Co-Authors: Cyprien Dulac, Van Trung Nguyen, Annemieke A Michels, Luigi Lania, Alessandro Fraldi, Francois Bonnet, Giuliana Napolitano, Olivier BensaudeAbstract:The positive transcription elongation factor (P-TEFb) comprises a kinase, CDK9, and a Cyclin T1 or T2. Its activity is inhibited by association with the HEXIM1 or HEXIM2 protein bound to 7SK small nuclear RNA. HEXIM1 and HEXIM2 were found to form stable homo- and hetero-oligomers. Using yeast two-hybrid and transfection assays, we have now shown that the C-terminal domains of HEXIM proteins directly interact with each other. Hydrodynamic parameters measured by glycerol gradient ultracentrifugation and gel-permeation chromatography demonstrate that both purified recombinant and cellular HEXIM1 proteins form highly anisotropic particles. Chemical cross-links suggest that HEXIM1 proteins form dimers. The multimeric nature of HEXIM1 is maintained in P-TEFb·HEXIM1·7SK RNA complexes. Multiple P-TEFb modules are found in the inactive P-TEFb·HEXIM1·7SK complexes. It is proposed that 7SK RNA binding to a HEXIM1 multimer promotes the simultaneous recruitment and hence inactivation of multiple P-TEFb units.
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maq1 and 7SK RNA interact with cdk9 cyclin t complexes in a transcription dependent manner
Molecular and Cellular Biology, 2003Co-Authors: Annemieke A Michels, Van Trung Nguyen, Luigi Lania, Alessandro Fraldi, Francois Bonnet, Valerie Labas, Mia Edwards, Olivier BensaudeAbstract:Phosphorylation of the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) is a critical step required for transcription elongation (7) and for recruitment of the machinery involved in pre-mRNA maturation (3, 26, 46). The CTD is unphosphorylated when RNAP II assembles onto promoters (RNAP IIA). A class of negative transcription factors including the 5,6-dichlorozo-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor and the negative elongation factor causes transcriptional arrest shortly after initiation, during which the polymerase may fall off (60). To release this block, the CTD must be phosphorylated (RNAP IIO) by positive transcription elongation factor b (P-TEFb), a protein complex that comprises cyclin-dependent kinase 9 (CDK9) and a cyclin (T1 or T2) (45). P-TEFb kinase activity is required for transcription of most class II genes (6). The human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter uses a unique mechanism: the level of proviral DNA transcription is determined by recruitment of P-TEFb to the TAR (transactivation response) element, an RNA stem-loop structure that forms at the 5′ end of the viral transcript (4, 38, 59, 66). The viral genome encodes a very potent transactivator of its own transcription, the Tat protein. The formation of a quateRNAry complex among CDK9, cyclin T1, Tat, and TAR RNA determines the recruitment of human P-TEFb to the transcription elongation complex and the efficient synthesis of long productive viral transcripts (15, 18, 30, 33, 44, 65). Binding of the 7SK small nuclear RNA (snRNA) to P-TEFb has recently been shown to be associated with the inhibition of CDK9 kinase activity (41, 62). Core P-TEFb is active, whereas the P-TEFb/7SK RNA complex is inactive. P-TEFb and 7SK associate in a reversible manner. Inhibition of cellular transcription by chemical agents or UV irradiation triggers the complete disruption of the P-TEFb/7SK complex and enhances CDK9 activity. In this study, we searched for additional cellular proteins that may be present in the P-TEFb/7SK RNA complex. A single novel P-TEFb subunit was found and termed MAQ1 (for menage a quatre), alluding to MAT1 (for menage a trois), which associates with CDK9-related CDK7 and cyclin H (10). The transcription-dependent interaction of P-TEFb with 7SK and MAQ1 may contribute to a feedback loop that modulates the activity of RNAP II.
