RESUMEN
The post-translational modification of key residues at the C-terminal domain of RNA polymerase II (RNAP2-CTD) coordinates transcription, splicing, and RNA processing by modulating its capacity to act as a landing platform for a variety of protein complexes. Here, we identify a new modification at the CTD, the deimination of arginine and its conversion to citrulline by peptidyl arginine deiminase 2 (PADI2), an enzyme that has been associated with several diseases, including cancer. We show that, among PADI family members, only PADI2 citrullinates R1810 (Cit1810) at repeat 31 of the CTD. Depletion of PADI2 or loss of R1810 results in accumulation of RNAP2 at transcription start sites, reduced gene expression, and inhibition of cell proliferation. Cit1810 is needed for interaction with the P-TEFb (positive transcription elongation factor b) kinase complex and for its recruitment to chromatin. In this way, CTD-Cit1810 favors RNAP2 pause release and efficient transcription in breast cancer cells.
Asunto(s)
Neoplasias de la Mama/enzimología , Procesamiento Proteico-Postraduccional , ARN Polimerasa II/metabolismo , Transcripción Genética , Arginina , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular , Citrulinación , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Dominios Proteicos , Arginina Deiminasa Proteína-Tipo 2 , Desiminasas de la Arginina Proteica/genética , Desiminasas de la Arginina Proteica/metabolismo , ARN Polimerasa II/química , ARN Polimerasa II/genética , Transducción de SeñalRESUMEN
The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is composed of a repetition of YSPTSPS heptads and functions as a loading platform for protein complexes that regulate transcription, splicing, and maturation of RNAs. Here, we studied mammalian CTD mutants to analyze the function of tyrosine1 residues in the transcription cycle. Mutation of 3/4 of the tyrosine residues (YFFF mutant) resulted in a massive read-through transcription phenotype in the antisense direction of promoters as well as in the 3' direction several hundred kilobases downstream of genes. The YFFF mutant shows reduced Pol II at promoter-proximal pause sites, a loss of interaction with the Mediator and Integrator complexes, and impaired recruitment of these complexes to chromatin. Consistent with these observations, Pol II loading at enhancers and maturation of snRNAs are altered in the YFFF context genome-wide. We conclude that tyrosine1 residues of the CTD control termination of transcription by Pol II.
Asunto(s)
ARN Polimerasa II/genética , ARN Mensajero/biosíntesis , Terminación de la Transcripción Genética/fisiología , Transcripción Genética/fisiología , Tirosina/genética , Línea Celular Tumoral , Cromatina/metabolismo , Humanos , Mutación/genética , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/metabolismo , ARN Nuclear Pequeño/genéticaRESUMEN
The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y1-S2-P3-T4-S5-P6-S7. Dynamic phosphorylation of the CTD coordinates Pol II progression through the transcription cycle. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y1, S2, T4, S5, and S7 in mammalian and yeast cells. Although specific phosphorylation signatures dominate, adjacent CTD repeats can be differently phosphorylated, leading to a high variation of coexisting phosphosites in mono- and di-heptad CTD repeats. Inhibition of CDK9 kinase specifically reduces S2 phosphorylation levels within the CTD.
Asunto(s)
ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/metabolismo , Humanos , Mamíferos , Espectrometría de Masas , Datos de Secuencia Molecular , Biblioteca de Péptidos , Fosforilación , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Secuencias Repetitivas de Aminoácido , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-ActividadRESUMEN
Low-complexity (LC) domains regulate the aggregation and phase transition of proteins in a modification-dependent manner. The study of LC domain modifications has now become feasible, as shown by genetic variants of the carboxy-terminal domain (CTD) of RNA Polymerase II (Pol II) that provide access to the type and position of modifications of a LC domain by mass spectrometry (MS).
Asunto(s)
Sustitución de Aminoácidos , Proteínas de Unión a Calmodulina/química , ARN Polimerasa II/química , Proteína FUS de Unión a ARN/química , Proteínas de Unión al ARN/química , Factores Asociados con la Proteína de Unión a TATA/química , Secuencia de Aminoácidos , Animales , Proteínas de Unión a Calmodulina/genética , Pollos , Expresión Génica , Humanos , Ratones , Mutación , Dominios Proteicos , ARN Polimerasa II/genética , Proteína EWS de Unión a ARN , Proteína FUS de Unión a ARN/genética , Proteínas de Unión al ARN/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores Asociados con la Proteína de Unión a TATA/genética , Transcripción GenéticaRESUMEN
Dynamic modification of heptad-repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 of RNA polymerase II (RNAPII) C-terminal domain (CTD) regulates transcription-coupled processes. Mass spectrometry analysis revealed that K7-residues in non-consensus repeats of human RNAPII are modified by acetylation, or mono-, di-, and tri-methylation. K7ac, K7me2, and K7me3 were found exclusively associated with phosphorylated CTD peptides, while K7me1 occurred also in non-phosphorylated CTD. The monoclonal antibody 1F5 recognizes K7me1/2 residues in CTD and reacts with RNAPIIA. Treatment of cellular extracts with phosphatase or of cells with the kinase inhibitor flavopiridol unmasked the K7me1/2 epitope in RNAPII0, consistent with the association of K7me1/2 marks with phosphorylated CTD peptides. Genome-wide profiling revealed high levels of K7me1/2 marks at the transcriptional start site of genes for sense and antisense transcribing RNAPII. The new K7 modifications further expand the mammalian CTD code to allow regulation of differential gene expression.
Asunto(s)
Lisina/metabolismo , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Acetilación , Anticuerpos Monoclonales/metabolismo , Línea Celular , Regulación de la Expresión Génica , Humanos , Espectrometría de Masas , Metilación , Modelos Moleculares , Estructura Terciaria de Proteína , ARN Polimerasa II/genética , Iniciación de la Transcripción GenéticaRESUMEN
In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. 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. Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers. 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. Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription, and Pol II stability.DOI: http://dx.doi.org/10.7554/eLife.02105.001.
Asunto(s)
Elementos sin Sentido (Genética) , Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Tirosina/metabolismo , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Humanos , Mutación , Fosforilación , ARN Polimerasa II/química , ARN Polimerasa II/genéticaRESUMEN
The carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) in mammals undergoes extensive posttranslational modification, which is essential for transcriptional initiation and elongation. Here, we show that the CTD of RNAPII is methylated at a single arginine (R1810) by the coactivator-associated arginine methyltransferase 1 (CARM1). Although methylation at R1810 is present on the hyperphosphorylated form of RNAPII in vivo, Ser2 or Ser5 phosphorylation inhibits CARM1 activity toward this site in vitro, suggesting that methylation occurs before transcription initiation. Mutation of R1810 results in the misexpression of a variety of small nuclear RNAs and small nucleolar RNAs, an effect that is also observed in Carm1(-/-) mouse embryo fibroblasts. These results demonstrate that CTD methylation facilitates the expression of select RNAs, perhaps serving to discriminate the RNAPII-associated machinery recruited to distinct gene types.