RESUMO
The RNA polymerase II carboxy-terminal domain (CTD) consists of conserved heptapeptide repeats that can be phosphorylated to influence distinct stages of the transcription cycle, including RNA processing. Although CTD-associated proteins have been identified, phospho-dependent CTD interactions have remained elusive. Proximity-dependent biotinylation (PDB) has recently emerged as an alternative approach to identify protein-protein associations in the native cellular environment. In this study, we present a PDB-based map of the fission yeast RNAPII CTD interactome in living cells and identify phospho-dependent CTD interactions by using a mutant in which Ser2 was replaced by alanine in every repeat of the fission yeast CTD. This approach revealed that CTD Ser2 phosphorylation is critical for the association between RNAPII and the histone methyltransferase Set2 during transcription elongation, but is not required for 3' end processing and transcription termination. Accordingly, loss of CTD Ser2 phosphorylation causes a global increase in antisense transcription, correlating with elevated histone acetylation in gene bodies. Our findings reveal that the fundamental role of CTD Ser2 phosphorylation is to establish a chromatin-based repressive state that prevents cryptic intragenic transcription initiation.
Assuntos
RNA Polimerase II , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Serina , Transcrição Gênica , RNA Polimerase II/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Fosforilação , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Serina/metabolismo , Histonas/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Regulação Fúngica da Expressão Gênica , RNA Antissenso/metabolismo , RNA Antissenso/genética , Domínios Proteicos , AcetilaçãoRESUMO
Quiescent cells require a continuous supply of proteins to maintain protein homeostasis. In fission yeast, entry into quiescence is triggered by nitrogen stress, leading to the inactivation of TORC1 and the activation of TORC2. Here, we report that the Greatwall-Endosulfine-PPA/B55 pathway connects the downregulation of TORC1 with the upregulation of TORC2, resulting in the activation of Elongator-dependent tRNA modifications essential for sustaining the translation programme during entry into quiescence. This process promotes U34 and A37 tRNA modifications at the anticodon stem loop, enhancing translation efficiency and fidelity of mRNAs enriched for AAA versus AAG lysine codons. Notably, some of these mRNAs encode inhibitors of TORC1, activators of TORC2, tRNA modifiers, and proteins necessary for telomeric and subtelomeric functions. Therefore, we propose a novel mechanism by which cells respond to nitrogen stress at the level of translation, involving a coordinated interplay between the tRNA epitranscriptome and biased codon usage.
RESUMO
The packaging of the genetic material into chromatin imposes the remodeling of this barrier to allow efficient transcription. RNA polymerase II activity is coupled with several histone modification complexes that enforce remodeling. How RNA polymerase III (Pol III) counteracts the inhibitory effect of chromatin is unknown. We report here a mechanism where RNA Polymerase II (Pol II) transcription is required to prime and maintain nucleosome depletion at Pol III loci and contributes to efficient Pol III recruitment upon re-initiation of growth from stationary phase in Fission yeast. The Pcr1 transcription factor participates in the recruitment of Pol II, which affects local histone occupancy through the associated SAGA complex and a Pol II phospho-S2 CTD / Mst2 pathway. These data expand the central role of Pol II in gene expression beyond mRNA synthesis.
Assuntos
Montagem e Desmontagem da Cromatina , RNA Polimerase II , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromatina/genética , Cromatina/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Transcrição GênicaRESUMO
The recent development of epitranscriptomics revealed a new fundamental layer of gene expression, but the mapping of most RNA modifications remains technically challenging. Here, we describe our protocol for Rho-Seq, which enables the mapping of dihydrouridine RNA modification at single-nucleotide resolution. Rho-Seq relies on specific rhodamine-labeling of a subset of modified nucleotides that hinders reverse transcription. Although Rho-Seq was initially applied to the detection of dihydrouridine, we show here that it is applicable to other modifications including 7-methylguanosine or 4-thiouridine. For complete details on the use and execution of this protocol, please refer to Finet et al. (2022).
Assuntos
Nucleotídeos , Tiouridina , RNA/genética , Rodaminas , Análise de Sequência de RNA/métodosRESUMO
The universal dihydrouridine (D) epitranscriptomic mark results from a reduction of uridine by the Dus family of NADPH-dependent reductases and is typically found within the eponym D-loop of tRNAs. Despite its apparent simplicity, D is structurally unique, with the potential to deeply affect the RNA backbone and many, if not all, RNA-connected processes. The first landscape of its occupancy within the tRNAome was reported 20 years ago. Its potential biological significance was highlighted by observations ranging from a strong bias in its ecological distribution to the predictive nature of Dus enzymes overexpression for worse cancer patient outcomes. The exquisite specificity of the Dus enzymes revealed by a structure-function analyses and accumulating clues that the D distribution may expand beyond tRNAs recently led to the development of new high-resolution mapping methods, including Rho-seq that established the presence of D within mRNAs and led to the demonstration of its critical physiological relevance.
Assuntos
Oxirredutases , RNA de Transferência , Humanos , Oxirredutases/genética , RNA/química , RNA Mensageiro/genética , RNA de Transferência/química , RNA de Transferência/genética , Uridina/químicaRESUMO
The epitranscriptome has emerged as a new fundamental layer of control of gene expression. Nevertheless, the determination of the transcriptome-wide occupancy and function of RNA modifications remains challenging. Here we have developed Rho-seq, an integrated pipeline detecting a range of modifications through differential modification-dependent rhodamine labeling. Using Rho-seq, we confirm that the reduction of uridine to dihydrouridine (D) by the Dus reductase enzymes targets tRNAs in E. coli and fission yeast. We find that the D modification is also present on fission yeast mRNAs, particularly those encoding cytoskeleton-related proteins, which is supported by large-scale proteome analyses and ribosome profiling. We show that the α-tubulin encoding mRNA nda2 undergoes Dus3-dependent dihydrouridylation, which affects its translation. The absence of the modification on nda2 mRNA strongly impacts meiotic chromosome segregation, resulting in low gamete viability. Applying Rho-seq to human cells revealed that tubulin mRNA dihydrouridylation is evolutionarily conserved.
Assuntos
Segregação de Cromossomos , Escherichia coli/genética , Meiose , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Schizosaccharomyces/genética , Uridina/metabolismo , Cromossomos Bacterianos , Cromossomos Fúngicos , Cromossomos Humanos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Células HCT116 , Humanos , Oxirredução , RNA Bacteriano/metabolismo , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Análise de Sequência de RNA , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
First identified 20 years ago as an RNA polymerase II-associated putative histone acetyltransferase, the conserved Elongator complex has since been recognized as the central player of a complex, regulated, and biologically relevant epitranscriptomic pathway targeting the wobble uridine of some tRNAs. Numerous studies have contributed to three emerging concepts resulting from anticodon modification by Elongator: the codon-specific control of translation, the ability of reprogramming translation in various physiological or pathological contexts, and the maintenance of proteome integrity by counteracting protein aggregation. These three aspects of tRNA modification by Elongator constitute a new layer of regulation that fundamentally contributes to gene expression and are now recognized as being critically involved in various human diseases.
RESUMO
Nutrient availability has a profound impact on cell fate. Upon nitrogen starvation, wild-type fission yeast cells uncouple cell growth from cell division to generate small, round-shaped cells that are competent for sexual differentiation. The TORC1 (TOR complex 1) and TORC2 complexes exert opposite controls on cell growth and cell differentiation, but little is known about how their activity is coordinated. We show that transfer RNA (tRNA) modifications by Elongator are critical for this regulation by promoting the translation of both key components of TORC2 and repressors of TORC1. We further identified the TORC2 pathway as an activator of Elongator by down-regulating a Gsk3 (glycogen synthase kinase 3)-dependent inhibitory phosphorylation of Elongator. Therefore, a feedback control is operating between TOR complex (TORC) signaling and tRNA modification by Elongator to enforce the advancement of mitosis that precedes cell differentiation.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Nutrientes/genética , Elongação Traducional da Cadeia Peptídica/genética , RNA de Transferência/genética , Schizosaccharomyces/genética , Transdução de Sinais/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Regulação Fúngica da Expressão Gênica/genética , Quinase 3 da Glicogênio Sintase/genética , Mitose/genética , Fosforilação/genéticaRESUMO
Background: The spliceosomal transfer of a short spliced leader (SL) RNA to an independent pre-mRNA molecule is called SL trans-splicing and is widespread in the nematode Caenorhabditis elegans. While RNA-sequencing (RNA-seq) data contain information on such events, properly documented methods to extract them are lacking. Findings: To address this, we developed SL-quant, a fast and flexible pipeline that adapts to paired-end and single-end RNA-seq data and accurately quantifies SL trans-splicing events. It is designed to work downstream of read mapping and uses the reads left unmapped as primary input. Briefly, the SL sequences are identified with high specificity and are trimmed from the input reads, which are then remapped on the reference genome and quantified at the nucleotide position level (SL trans-splice sites) or at the gene level. Conclusions: SL-quant completes within 10 minutes on a basic desktop computer for typical C. elegans RNA-seq datasets and can be applied to other species as well. Validating the method, the SL trans-splice sites identified display the expected consensus sequence, and the results of the gene-level quantification are predictive of the gene position within operons. We also compared SL-quant to a recently published SL-containing read identification strategy that was found to be more sensitive but less specific than SL-quant. Both methods are implemented as a bash script available under the MIT license [1]. Full instructions for its installation, usage, and adaptation to other organisms are provided.
Assuntos
Caenorhabditis elegans/genética , Biologia Computacional/métodos , Splicing de RNA , RNA Líder para Processamento , Análise de Sequência de RNA/métodos , Trans-Splicing , Algoritmos , Animais , Genoma , Genômica/métodos , Óperon , Precursores de RNA , RNA Mensageiro/genética , SoftwareRESUMO
Antisense (as)lncRNAs can regulate gene expression but the underlying mechanisms and the different cofactors involved remain unclear. Using Native Elongating Transcript sequencing, here we show that stabilization of antisense Exo2-sensitivite lncRNAs (XUTs) results in the attenuation, at the nascent transcription level, of a subset of highly expressed genes displaying prominent promoter-proximal nucleosome depletion and histone acetylation. Mechanistic investigations on the catalase gene ctt1 revealed that its induction following oxidative stress is impaired in Exo2-deficient cells, correlating with the accumulation of an asXUT. Interestingly, expression of this asXUT was also activated in wild-type cells upon oxidative stress, concomitant to ctt1 induction, indicating a potential attenuation feedback. This attenuation correlates with asXUT abundance, it is transcriptional, characterized by low RNAPII-ser5 phosphorylation, and it requires an histone deacetylase activity and the conserved Set2 histone methyltransferase. Finally, we identified Dicer as another RNA processing factor acting on ctt1 induction, but independently of Exo2. We propose that asXUTs could modulate the expression of their paired-sense genes when it exceeds a critical threshold, using a conserved mechanism independent of RNAi.
Assuntos
Regulação Fúngica da Expressão Gênica , RNA Antissenso/metabolismo , RNA Fúngico/metabolismo , RNA Longo não Codificante/metabolismo , Schizosaccharomyces/genética , Acetilação , Catalase/genética , Endorribonucleases/metabolismo , Exodesoxirribonucleases/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Estresse Oxidativo/genética , Regiões Promotoras Genéticas , Interferência de RNA , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transcrição Gênica/genéticaRESUMO
The distribution of modified histones within the fission yeast Schizosaccharomyces pombe genome is ultimately dependent upon the transcriptional activity and in turn influences the ability of the polymerases to bind and progress through the chromatin template. The Chromatin Immunoprecipitation-Polymerase Chain Reaction (ChIP-PCR) method currently provides the highest resolution, accuracy, and reproducibility to characterize histones modifications within a defined region of the genome. The following protocol details the method applied to S. pombe.
Assuntos
Imunoprecipitação da Cromatina/métodos , Metilação de DNA/fisiologia , Genoma Fúngico , Reação em Cadeia da Polimerase/métodos , Schizosaccharomyces , Ubiquitinação/fisiologia , Acetilação , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMO
The cell fate decision leading to gametogenesis requires the convergence of multiple signals on the promoter of a master regulator. In fission yeast, starvation-induced signaling leads to the transcriptional induction of the ste11 gene, which encodes the central inducer of mating and gametogenesis, known as sporulation. We find that the long intergenic non-coding (linc) RNA rse1 is transcribed divergently upstream of the ste11 gene. During vegetative growth, rse1 directly recruits a Mug187-Lid2-Set1 complex that mediates cis repression at the ste11 promoter through SET3C-dependent histone deacetylation. The absence of rse1 bypasses the starvation-induced signaling and induces gametogenesis in the presence of nutrients. Our data reveal that the remodeling of chromatin through ncRNA scaffolding of repressive complexes that is observed in higher eukaryotes is a conserved, likely very ancient mechanism for tight control of cell differentiation.
Assuntos
RNA Fúngico/metabolismo , RNA Longo não Codificante/metabolismo , Schizosaccharomyces/fisiologia , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Antisense transcription can regulate sense gene expression. However, previous annotations of antisense transcription units have been based on detection of mature antisense long noncoding (aslnc)RNAs by RNA-seq and/or microarrays, only giving a partial view of the antisense transcription landscape and incomplete molecular bases for antisense-mediated regulation. Here, we used native elongating transcript sequencing to map genome-wide nascent antisense transcription in fission yeast. Strikingly, antisense transcription was detected for most protein-coding genes, correlating with low sense transcription, especially when overlapping the mRNA start site. RNA profiling revealed that the resulting aslncRNAs mainly correspond to cryptic Xrn1/Exo2-sensitive transcripts (XUTs). ChIP-seq analyses showed that antisense (as)XUT's expression is associated with specific histone modification patterns. Finally, we showed that asXUTs are controlled by the histone chaperone Spt6 and respond to meiosis induction, in both cases anti-correlating with levels of the paired-sense mRNAs, supporting physiological significance to antisense-mediated gene attenuation. Our work highlights that antisense transcription is much more extended than anticipated and might constitute an additional nonpromoter determinant of gene regulation complexity.
Assuntos
Regulação Fúngica da Expressão Gênica , RNA Antissenso/biossíntese , Schizosaccharomyces/genética , Transcrição Gênica , Chaperonas de Histonas/metabolismo , Código das Histonas , Meiose/genética , Elongação Traducional da Cadeia Peptídica , Interferência de RNA , Estabilidade de RNA , RNA Antissenso/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Análise de Sequência de RNARESUMO
Some long noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (RNAPII) are retained on chromatin, where they regulate RNAi and chromatin structure. The molecular basis of this retention remains unknown. We show that in fission yeast serine 7 (Ser7) of the C-terminal domain (CTD) of RNAPII is required for efficient siRNA generation for RNAi-dependent heterochromatin formation. Surprisingly, Ser7 facilitates chromatin retention of nascent heterochromatic RNAs (hRNAs). Chromatin retention of hRNAs and siRNA generation requires both Ser7 and an RNA-binding activity of the chromodomain of Chp1, a subunit of the RNA-induced transcriptional silencing (RITS) complex. Furthermore, RITS associates with RNAPII in a Ser7-dependent manner. We propose that Ser7 promotes cotranscriptional chromatin retention of hRNA by recruiting the RNA-chromatin connector protein Chp1, which facilitates RNAi-dependent heterochromatin formation. Our findings reveal a function of the CTD code: linking ncRNA transcription to RNAi for heterochromatin formation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Heterocromatina/metabolismo , RNA Polimerase II/metabolismo , RNA Fúngico/metabolismo , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Heterocromatina/genética , Domínios Proteicos , RNA Polimerase II/genética , RNA Fúngico/genética , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Serina/genética , Serina/metabolismoRESUMO
The occupancy of nucleosomes governs access to the eukaryotic genomes and results from a combination of biophysical features and the effect of ATP-dependent remodelling complexes. Most promoter regions show a conserved pattern characterized by a nucleosome-depleted region (NDR) flanked by nucleosomal arrays. The conserved RSC remodeler was reported to be critical to establish NDR in vivo in budding yeast but other evidences suggested that this activity may not be conserved in fission yeast. By reanalysing and expanding previously published data, we propose that NDR formation requires, at least partially, RSC in both yeast species. We also discuss the most prominent biological role of RSC and the possibility that non-essential subunits do not define alternate versions of the complex.
Assuntos
Adenosina Trifosfatases/genética , Montagem e Desmontagem da Cromatina/genética , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/genética , Adenosina Trifosfatases/metabolismo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Especificidade da EspécieRESUMO
In fission yeast, the ste11 gene encodes the master regulator initiating the switch from vegetative growth to gametogenesis. In a previous paper, we showed that the methylation of H3K4 and consequent promoter nucleosome deacetylation repress ste11 induction and cell differentiation (Materne et al., 2015) but the regulatory steps remain poorly understood. Here we report a genetic screen that highlighted H2B deubiquitylation and the RSC remodeling complex as activators of ste11 expression. Mechanistic analyses revealed more complex, opposite roles of H2Bubi at the promoter where it represses expression, and over the transcribed region where it sustains it. By promoting H3K4 methylation at the promoter, H2Bubi initiates the deacetylation process, which decreases chromatin remodeling by RSC. Upon induction, this process is reversed and efficient NDR (nucleosome depleted region) formation leads to high expression. Therefore, H2Bubi represses gametogenesis by opposing the recruitment of RSC at the promoter of the master regulator ste11 gene.
Assuntos
Montagem e Desmontagem da Cromatina , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Fatores de Transcrição/antagonistas & inibidores , UbiquitinaçãoRESUMO
Tumor initiation in the intestine can rapidly occur from Lgr5(+) crypt columnar stem cells. Dclk1 is a marker of differentiated Tuft cells and, when coexpressed with Lgr5, also marks intestinal cancer stem cells. Here, we show that Elp3, the catalytic subunit of the Elongator complex, is required for Wnt-driven intestinal tumor initiation and radiation-induced regeneration by maintaining a subpool of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Elp3 deficiency dramatically delayed tumor appearance in Apc-mutated intestinal epithelia and greatly prolonged mice survival without affecting the normal epithelium. Specific ablation of Elp3 in Lgr5(+) cells resulted in marked reduction of polyp formation upon Apc inactivation, in part due to a decreased number of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Mechanistically, Elp3 is induced by Wnt signaling and promotes Sox9 translation, which is needed to maintain the subpool of Lgr5(+)/Dclk1(+) cancer stem cells. Consequently, Elp3 or Sox9 depletion led to similar defects in Dclk1(+) cancer stem cells in ex vivo organoids. Finally, Elp3 deficiency strongly impaired radiation-induced intestinal regeneration, in part because of decreased Sox9 protein levels. Together, our data demonstrate the crucial role of Elp3 in maintaining a subpopulation of Lgr5-derived and Sox9-expressing cells needed to trigger Wnt-driven tumor initiation in the intestine.
Assuntos
Histona Acetiltransferases/metabolismo , Intestinos/fisiopatologia , Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Regeneração/fisiologia , Proteínas Wnt/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Quinases Semelhantes a Duplacortina , Expressão Gênica/efeitos da radiação , Células HCT116 , Células HEK293 , Células HT29 , Histona Acetiltransferases/genética , Humanos , Mucosa Intestinal/metabolismo , Intestinos/efeitos da radiação , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Neoplasias/genética , Neoplasias/patologia , Proteínas do Tecido Nervoso/genética , Técnicas de Cultura de Órgãos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Regeneração/genética , Regeneração/efeitos da radiação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Proteínas Wnt/genéticaRESUMO
The phosphorylation of the RNA polymerase II C-terminal domain (CTD) plays a key role in delineating transcribed regions within chromatin by recruiting histone methylases and deacetylases. Using genome-wide nucleosome mapping, we show that CTD S2 phosphorylation controls nucleosome dynamics in the promoter of a subset of 324 genes, including the regulators of cell differentiation ste11 and metabolic adaptation inv1. Mechanistic studies on these genes indicate that during gene activation a local increase of phospho-S2 CTD nearby the promoter impairs the phospho-S5 CTD-dependent recruitment of Set1 and the subsequent recruitment of specific HDACs, which leads to nucleosome depletion and efficient transcription. The early increase of phospho-S2 results from the phosphorylation of the CTD S2 kinase Lsk1 by MAP kinase in response to cellular signalling. The artificial tethering of the Lsk1 kinase at the ste11 promoter is sufficient to activate transcription. Therefore, signalling through the CTD code regulates promoter nucleosomes dynamics.
Assuntos
Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , RNA Polimerase II/metabolismo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Fosforilação , Regiões Promotoras Genéticas , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Análise de Sequência de DNA , Transdução de Sinais , Transcrição Gênica , Ativação TranscricionalRESUMO
Cyclin-dependent kinase (Cdk) activation and RNA polymerase II transcription are linked by the Cdk7 kinase, which phosphorylates Cdks as a trimeric Cdk-activating kinase (CAK) complex, and serine 5 within the polymerase II (Pol II) C-terminal domain (CTD) as transcription factor TFIIH-bound CAK. However, the physiological importance of integrating these processes is not understood. Besides the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. The two enzymes have been proposed to act redundantly to activate Cdc2. Using an improved analogue-sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20-amino-acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through its well-described RNA Pol II CTD kinase activity and also through the Cdc2-activating kinase activity.