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Integrator complex regulates NELF-mediated RNA polymerase II pause/release and processivity at coding genes.

Stadelmayer B, Micas G, Gamot A, Martin P, Malirat N, Koval S, Raffel R, Sobhian B, Severac D, Rialle S, Parrinello H, Cuvier O, Benkirane M - Nat Commun (2014)

Bottom Line: The strength of RNAPII pausing is determined by the nature of the NELF-associated INTScom subunits.Interestingly, in addition to controlling RNAPII pause-release INTS11 catalytic subunit of the INTScom is required for RNAPII processivity.Revealing these unexpected functions of INTScom in regulating RNAPII pause-release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Human Genetics, CNRS UPR1142, Laboratory of Molecular Virology; MGX-Montpellier GenomiX, 141 rue de la Cardonille, Montpellier 34396, France [2] LBME-CNRS, Cell Cycle Chromatin Dynamics Laboratory. University Paul Sabatier, Toulouse 31061, France [3] INRA, TOXALIM (Research Centre in Food Toxicology), Toulouse 31300, France [4] IGF, MGX-Montpellier GenomiX, France.

ABSTRACT
RNA polymerase II (RNAPII) pausing/termination shortly after initiation is a hallmark of gene regulation. Here, we show that negative elongation factor (NELF) interacts with Integrator complex subunits (INTScom), RNAPII and Spt5. The interaction between NELF and INTScom subunits is RNA and DNA independent. Using both human immunodeficiency virus type 1 promoter and genome-wide analyses, we demonstrate that Integrator subunits specifically control NELF-mediated RNAPII pause/release at coding genes. The strength of RNAPII pausing is determined by the nature of the NELF-associated INTScom subunits. Interestingly, in addition to controlling RNAPII pause-release INTS11 catalytic subunit of the INTScom is required for RNAPII processivity. Finally, INTScom target genes are enriched in human immunodeficiency virus type 1 transactivation response element/NELF binding element and in a 3' box sequence required for small nuclear RNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNAPII pause-release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle.

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Immuno-purification of NELF.(a) Flag/HA-epitope-tagged NELF-E (eNELF-E) from HeLa S3 Dignam nuclear extracts was sequentially immunopurified on anti-Flag and anti-HA antibody-conjugated agarose beads. Purified material was separated by SDS–PAGE and visualised by silver staining. eNELF-E-associated proteins were identified by MS (see Supplementary Dataset 1). (b) Flag/HA IPs from samples shown in (a) were separated by SDS–PAGE and the presence of eNELF-E-associated proteins identified was confirmed by immunoblotting. (c) Glycerol gradient sedimentation analysis of eNELF-E. Flag-purified eNELF-E-associated complexes were separated by centrifugation through a 12–40% glycerol gradient. Material of even-numbered fractions was resolved by SDS–PAGE and probed for identified proteins. (d) Reciprocal IPs (ReIPs): Flag-purified eNELF-E (Input) was subjected to IP using anti-Spt5, anti-INTS13, anti-HA antibodies or irrelevant rabbit IgG (IPr) or mouse IgG (IPm). Input, IP, as well as flow through (FT) were probed for eNELF-associated proteins.
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f1: Immuno-purification of NELF.(a) Flag/HA-epitope-tagged NELF-E (eNELF-E) from HeLa S3 Dignam nuclear extracts was sequentially immunopurified on anti-Flag and anti-HA antibody-conjugated agarose beads. Purified material was separated by SDS–PAGE and visualised by silver staining. eNELF-E-associated proteins were identified by MS (see Supplementary Dataset 1). (b) Flag/HA IPs from samples shown in (a) were separated by SDS–PAGE and the presence of eNELF-E-associated proteins identified was confirmed by immunoblotting. (c) Glycerol gradient sedimentation analysis of eNELF-E. Flag-purified eNELF-E-associated complexes were separated by centrifugation through a 12–40% glycerol gradient. Material of even-numbered fractions was resolved by SDS–PAGE and probed for identified proteins. (d) Reciprocal IPs (ReIPs): Flag-purified eNELF-E (Input) was subjected to IP using anti-Spt5, anti-INTS13, anti-HA antibodies or irrelevant rabbit IgG (IPr) or mouse IgG (IPm). Input, IP, as well as flow through (FT) were probed for eNELF-associated proteins.

Mentions: To gain more insight into NELF-mediated RNAPII pausing we immunopurified (IP) NELF and its associated partners from HeLa nuclear extracts. Dignam nuclear extracts prepared from control and Flag-HA-tagged NELF-E subunit (eNELF-E) expressing cells were subjected to tandem affinity chromatography as previously described16. Flag-HA IPed materials were run on SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and proteins were visualised by silver staining and identified by mass spectrometry (MS) (Fig. 1a and Supplementary Dataset 1). Major MS-identified eNELF-E nuclear partners are the core subunits of the NELF complex (A, B, C/D). As previously reported, both Spt5 and subunits of RNAPII were recovered4. Interestingly, MS identified all subunits of the Integrator complex (INTScom) (Fig. 1a). We first confirmed the interaction between INTScom subunits and NELF-E (Fig. 1b). As negative controls, subunits of the NURD and INO80 complexes were absent in eNELF-E immunopurified materials (Supplementary Fig. 1A). Interestingly, we found that RPB1 associated with eNELF-E is phosphorylated on Ser7 but not on Serine 2 or Ser5 (Fig. 1b and Supplementary Fig. 1A). We next asked whether the interaction between NELF and INTScom subunits is mediated through nucleic acids or by protein–protein interactions. For this purpose, nuclear extracts were treated with either RNase or DNase and Ethidium bromide prior IP using anti-Flag antibody. The presence of INTS3 and INTS11 subunits in IPed material was assessed by western blotting. We found that NELF/INTScom interaction is independent of RNA and DNA (Supplementary Fig. 1B). As positive control, RNase treatment resulted in reduced interaction between CyclinT1, CDK9 and HEXIM (Supplementary Fig. 1C). Thus, and in agreement with the recent finding by Yamaguchi and colleagues17, NELF/INTScom interaction is mediated through protein–protein interaction. To test whether NELF, INTScom, Spt5 and RPB1 associate in a single complex, glycerol gradient sedimentation of Flag-purified eNELF-E was performed (Fig. 1c). The presence of eNELF-E-interacting proteins in the collected fractions was analysed by western blot. Fig. 1c shows a major peak containing NELF and the INTS3 subunit (fraction 4) and a second peak containing NELF, INTScom and RPB1 (Fraction 16). To further characterise NELF-associated proteins, Flag IPed NELF-E was subjected to second IPs (ReIP) using anti-Spt5, anti-INTS13 and anti-HA antibodies. As shown in Fig. 1d, a small fraction of subunits of the INTScom, NELF-A, NELF-E, Spt5 and RPB1 were present in ReIP using anti-Spt5 and anti-INTS13 antibodies suggesting that a fraction of NELF associates with INTScom, possibly as part of a RNAPII complex on the gene. Altogether, these experiments suggest that NELF interacts with INTScom and with INTS3 independently of other INSTcom subunits.


Integrator complex regulates NELF-mediated RNA polymerase II pause/release and processivity at coding genes.

Stadelmayer B, Micas G, Gamot A, Martin P, Malirat N, Koval S, Raffel R, Sobhian B, Severac D, Rialle S, Parrinello H, Cuvier O, Benkirane M - Nat Commun (2014)

Immuno-purification of NELF.(a) Flag/HA-epitope-tagged NELF-E (eNELF-E) from HeLa S3 Dignam nuclear extracts was sequentially immunopurified on anti-Flag and anti-HA antibody-conjugated agarose beads. Purified material was separated by SDS–PAGE and visualised by silver staining. eNELF-E-associated proteins were identified by MS (see Supplementary Dataset 1). (b) Flag/HA IPs from samples shown in (a) were separated by SDS–PAGE and the presence of eNELF-E-associated proteins identified was confirmed by immunoblotting. (c) Glycerol gradient sedimentation analysis of eNELF-E. Flag-purified eNELF-E-associated complexes were separated by centrifugation through a 12–40% glycerol gradient. Material of even-numbered fractions was resolved by SDS–PAGE and probed for identified proteins. (d) Reciprocal IPs (ReIPs): Flag-purified eNELF-E (Input) was subjected to IP using anti-Spt5, anti-INTS13, anti-HA antibodies or irrelevant rabbit IgG (IPr) or mouse IgG (IPm). Input, IP, as well as flow through (FT) were probed for eNELF-associated proteins.
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Related In: Results  -  Collection

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f1: Immuno-purification of NELF.(a) Flag/HA-epitope-tagged NELF-E (eNELF-E) from HeLa S3 Dignam nuclear extracts was sequentially immunopurified on anti-Flag and anti-HA antibody-conjugated agarose beads. Purified material was separated by SDS–PAGE and visualised by silver staining. eNELF-E-associated proteins were identified by MS (see Supplementary Dataset 1). (b) Flag/HA IPs from samples shown in (a) were separated by SDS–PAGE and the presence of eNELF-E-associated proteins identified was confirmed by immunoblotting. (c) Glycerol gradient sedimentation analysis of eNELF-E. Flag-purified eNELF-E-associated complexes were separated by centrifugation through a 12–40% glycerol gradient. Material of even-numbered fractions was resolved by SDS–PAGE and probed for identified proteins. (d) Reciprocal IPs (ReIPs): Flag-purified eNELF-E (Input) was subjected to IP using anti-Spt5, anti-INTS13, anti-HA antibodies or irrelevant rabbit IgG (IPr) or mouse IgG (IPm). Input, IP, as well as flow through (FT) were probed for eNELF-associated proteins.
Mentions: To gain more insight into NELF-mediated RNAPII pausing we immunopurified (IP) NELF and its associated partners from HeLa nuclear extracts. Dignam nuclear extracts prepared from control and Flag-HA-tagged NELF-E subunit (eNELF-E) expressing cells were subjected to tandem affinity chromatography as previously described16. Flag-HA IPed materials were run on SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and proteins were visualised by silver staining and identified by mass spectrometry (MS) (Fig. 1a and Supplementary Dataset 1). Major MS-identified eNELF-E nuclear partners are the core subunits of the NELF complex (A, B, C/D). As previously reported, both Spt5 and subunits of RNAPII were recovered4. Interestingly, MS identified all subunits of the Integrator complex (INTScom) (Fig. 1a). We first confirmed the interaction between INTScom subunits and NELF-E (Fig. 1b). As negative controls, subunits of the NURD and INO80 complexes were absent in eNELF-E immunopurified materials (Supplementary Fig. 1A). Interestingly, we found that RPB1 associated with eNELF-E is phosphorylated on Ser7 but not on Serine 2 or Ser5 (Fig. 1b and Supplementary Fig. 1A). We next asked whether the interaction between NELF and INTScom subunits is mediated through nucleic acids or by protein–protein interactions. For this purpose, nuclear extracts were treated with either RNase or DNase and Ethidium bromide prior IP using anti-Flag antibody. The presence of INTS3 and INTS11 subunits in IPed material was assessed by western blotting. We found that NELF/INTScom interaction is independent of RNA and DNA (Supplementary Fig. 1B). As positive control, RNase treatment resulted in reduced interaction between CyclinT1, CDK9 and HEXIM (Supplementary Fig. 1C). Thus, and in agreement with the recent finding by Yamaguchi and colleagues17, NELF/INTScom interaction is mediated through protein–protein interaction. To test whether NELF, INTScom, Spt5 and RPB1 associate in a single complex, glycerol gradient sedimentation of Flag-purified eNELF-E was performed (Fig. 1c). The presence of eNELF-E-interacting proteins in the collected fractions was analysed by western blot. Fig. 1c shows a major peak containing NELF and the INTS3 subunit (fraction 4) and a second peak containing NELF, INTScom and RPB1 (Fraction 16). To further characterise NELF-associated proteins, Flag IPed NELF-E was subjected to second IPs (ReIP) using anti-Spt5, anti-INTS13 and anti-HA antibodies. As shown in Fig. 1d, a small fraction of subunits of the INTScom, NELF-A, NELF-E, Spt5 and RPB1 were present in ReIP using anti-Spt5 and anti-INTS13 antibodies suggesting that a fraction of NELF associates with INTScom, possibly as part of a RNAPII complex on the gene. Altogether, these experiments suggest that NELF interacts with INTScom and with INTS3 independently of other INSTcom subunits.

Bottom Line: The strength of RNAPII pausing is determined by the nature of the NELF-associated INTScom subunits.Interestingly, in addition to controlling RNAPII pause-release INTS11 catalytic subunit of the INTScom is required for RNAPII processivity.Revealing these unexpected functions of INTScom in regulating RNAPII pause-release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Human Genetics, CNRS UPR1142, Laboratory of Molecular Virology; MGX-Montpellier GenomiX, 141 rue de la Cardonille, Montpellier 34396, France [2] LBME-CNRS, Cell Cycle Chromatin Dynamics Laboratory. University Paul Sabatier, Toulouse 31061, France [3] INRA, TOXALIM (Research Centre in Food Toxicology), Toulouse 31300, France [4] IGF, MGX-Montpellier GenomiX, France.

ABSTRACT
RNA polymerase II (RNAPII) pausing/termination shortly after initiation is a hallmark of gene regulation. Here, we show that negative elongation factor (NELF) interacts with Integrator complex subunits (INTScom), RNAPII and Spt5. The interaction between NELF and INTScom subunits is RNA and DNA independent. Using both human immunodeficiency virus type 1 promoter and genome-wide analyses, we demonstrate that Integrator subunits specifically control NELF-mediated RNAPII pause/release at coding genes. The strength of RNAPII pausing is determined by the nature of the NELF-associated INTScom subunits. Interestingly, in addition to controlling RNAPII pause-release INTS11 catalytic subunit of the INTScom is required for RNAPII processivity. Finally, INTScom target genes are enriched in human immunodeficiency virus type 1 transactivation response element/NELF binding element and in a 3' box sequence required for small nuclear RNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNAPII pause-release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle.

Show MeSH
Related in: MedlinePlus