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Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells.

Descostes N, Heidemann M, Spinelli L, Schüller R, Maqbool MA, Fenouil R, Koch F, Innocenti C, Gut M, Gut I, Eick D, Andrau JC - Elife (2014)

Bottom Line: Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation.Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of promoter (5' associated) Pol II in mammalian cells, in contrast to what was described in yeast.Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the truncated Pol IIB form, and results in a lethal phenotype.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy, Université Aix-Marseille, Marseille, France Centre National de la Recherche Scientifique (CNRS) UMR6102, Marseille, France Inserm U631, Marseille, France Institut de Génétique Moléculaire de Montpellier (IGMM), CNRS-UMR5535, Montpellier, France.

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Sequence of the CTD heptads for the Tyr1 to Phe mutant (Y1F).Amino-acid composition of the C-terminal domain of the Y1F mutant (as described in the ‘Materials and methods–Construction of the CTD Y1F mutant’) used for phenotypic and western blot analyses (Figure 1).DOI:http://dx.doi.org/10.7554/eLife.02105.005
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fig1s2: Sequence of the CTD heptads for the Tyr1 to Phe mutant (Y1F).Amino-acid composition of the C-terminal domain of the Y1F mutant (as described in the ‘Materials and methods–Construction of the CTD Y1F mutant’) used for phenotypic and western blot analyses (Figure 1).DOI:http://dx.doi.org/10.7554/eLife.02105.005

Mentions: To analyze expression and pattern of Tyr1P modified Pol II, we took advantage of our previously developed Tyr1P specific antibodies (3D12) (Mayer et al., 2012). We investigated various mouse and human cells and could detect Tyr1P in western blots for all examined lines, in most cases associated with the hyper-phosphorylated IIO form of Pol II (Figure 1—figure supplement 1). To address the function of Tyr1P, we next generated Raji cell lines expressing Pol II resistant to α-amanitin (Chapman et al., 2004) and carrying either wild-type (WT) or a mutant Rpb1 gene with substitution of tyrosine to phenylalanine (Y1F) in CTD repeats 4 to 51 (Figure 1—figure supplement 2). After expression of the mutant, we observed that Y1F yielded a truncated Rpb1 (Pol IIB, Figure 1A) and was unable to form the hyper-phosphorylated IIO Pol II. After disruption of the activity of endogenous Pol II by α-amanitin (Figure 1B) and soon after disappearance of WT Rpb1, cells became rapidly inviable. This phenotype reveals an essential function of the Y1 residue that appears more drastic than T4A or S7A mutations, but comparable with that of S5A (Chapman et al., 2007; Hintermair et al., 2012). We conclude that Tyr1P very likely contributes to stabilization of CTD and may occur early within the transcription cycle.10.7554/eLife.02105.003Figure 1.Y1F mutations of the CTD heptads yield a truncated Pol IIB Rpb1.


Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells.

Descostes N, Heidemann M, Spinelli L, Schüller R, Maqbool MA, Fenouil R, Koch F, Innocenti C, Gut M, Gut I, Eick D, Andrau JC - Elife (2014)

Sequence of the CTD heptads for the Tyr1 to Phe mutant (Y1F).Amino-acid composition of the C-terminal domain of the Y1F mutant (as described in the ‘Materials and methods–Construction of the CTD Y1F mutant’) used for phenotypic and western blot analyses (Figure 1).DOI:http://dx.doi.org/10.7554/eLife.02105.005
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4042876&req=5

fig1s2: Sequence of the CTD heptads for the Tyr1 to Phe mutant (Y1F).Amino-acid composition of the C-terminal domain of the Y1F mutant (as described in the ‘Materials and methods–Construction of the CTD Y1F mutant’) used for phenotypic and western blot analyses (Figure 1).DOI:http://dx.doi.org/10.7554/eLife.02105.005
Mentions: To analyze expression and pattern of Tyr1P modified Pol II, we took advantage of our previously developed Tyr1P specific antibodies (3D12) (Mayer et al., 2012). We investigated various mouse and human cells and could detect Tyr1P in western blots for all examined lines, in most cases associated with the hyper-phosphorylated IIO form of Pol II (Figure 1—figure supplement 1). To address the function of Tyr1P, we next generated Raji cell lines expressing Pol II resistant to α-amanitin (Chapman et al., 2004) and carrying either wild-type (WT) or a mutant Rpb1 gene with substitution of tyrosine to phenylalanine (Y1F) in CTD repeats 4 to 51 (Figure 1—figure supplement 2). After expression of the mutant, we observed that Y1F yielded a truncated Rpb1 (Pol IIB, Figure 1A) and was unable to form the hyper-phosphorylated IIO Pol II. After disruption of the activity of endogenous Pol II by α-amanitin (Figure 1B) and soon after disappearance of WT Rpb1, cells became rapidly inviable. This phenotype reveals an essential function of the Y1 residue that appears more drastic than T4A or S7A mutations, but comparable with that of S5A (Chapman et al., 2007; Hintermair et al., 2012). We conclude that Tyr1P very likely contributes to stabilization of CTD and may occur early within the transcription cycle.10.7554/eLife.02105.003Figure 1.Y1F mutations of the CTD heptads yield a truncated Pol IIB Rpb1.

Bottom Line: Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation.Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of promoter (5' associated) Pol II in mammalian cells, in contrast to what was described in yeast.Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the truncated Pol IIB form, and results in a lethal phenotype.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy, Université Aix-Marseille, Marseille, France Centre National de la Recherche Scientifique (CNRS) UMR6102, Marseille, France Inserm U631, Marseille, France Institut de Génétique Moléculaire de Montpellier (IGMM), CNRS-UMR5535, Montpellier, France.

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