RESUMEN
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.
Asunto(s)
ARN Polimerasa II , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Serina , Transcripción Genética , ARN Polimerasa II/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Fosforilación , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Serina/metabolismo , Histonas/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Regulación Fúngica de la Expresión Génica , ARN sin Sentido/metabolismo , ARN sin Sentido/genética , Dominios Proteicos , AcetilaciónRESUMEN
Eukaryotic genes must be condensed into chromatin while remaining accessible to the transcriptional machinery to support gene expression. Among the three eukaryotic RNA polymerases (RNAP), RNAPII is unique, partly because of the C-terminal domain (CTD) of its largest subunit, Rpb1. Rpb1 CTD can be extensively modified during the transcription cycle, allowing for the co-transcriptional recruitment of specific interacting proteins. These include chromatin remodeling factors that control the opening or closing of chromatin. How the CTD-less RNAPI and RNAPIII deal with chromatin at rRNA and tRNA genes is less understood. Here, we review recent advances in our understanding of how the chromatin at tRNA genes and rRNA genes can be remodeled in response to environmental cues in yeast, with a particular focus on the role of local RNAPII transcription in recruiting chromatin remodelers at these loci. In fission yeast, RNAPII transcription at tRNA genes is important to re-establish a chromatin environment permissive to tRNA transcription, which supports growth from stationary phase. In contrast, local RNAPII transcription at rRNA genes correlates with the closing of the chromatin in starvation in budding and fission yeast, suggesting a role in establishing silent chromatin. These opposite roles might support a general model where RNAPII transcription recruits chromatin remodelers to tRNA and rRNA genes to promote the closing and reopening of chromatin in response to the environment.
Asunto(s)
ARN Polimerasa II , Schizosaccharomyces , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Cromatina/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Transcripción Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN de Transferencia/genéticaRESUMEN
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.
Asunto(s)
Ensamble y Desensamble de Cromatina , ARN Polimerasa II , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromatina/genética , Cromatina/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Transcripción GenéticaRESUMEN
Transcription by RNA polymerase I (RNAPI) represents most of the transcriptional activity in eukaryotic cells and is associated with the production of mature ribosomal RNA (rRNA). As several rRNA maturation steps are coupled to RNAPI transcription, the rate of RNAPI elongation directly influences processing of nascent pre-rRNA, and changes in RNAPI transcription rate can result in alternative rRNA processing pathways in response to growth conditions and stress. However, factors and mechanisms that control RNAPI progression by influencing transcription elongation rate remain poorly understood. We show here that the conserved fission yeast RNA-binding protein Seb1 associates with the RNAPI transcription machinery and promotes RNAPI pausing states along the rDNA. The overall faster progression of RNAPI at the rDNA in Seb1-deficient cells impaired cotranscriptional pre-rRNA processing and the production of mature rRNAs. Given that Seb1 also influences pre-mRNA processing by modulating RNAPII progression, our findings unveil Seb1 as a pause-promoting factor for RNA polymerases I and II to control cotranscriptional RNA processing.
Asunto(s)
ARN Polimerasa I , Schizosaccharomyces , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Transcripción Genética , Procesamiento Postranscripcional del ARN , ADN Ribosómico/metabolismo , Schizosaccharomyces/genéticaRESUMEN
Intron retention is a type of alternative splicing where one or more introns remain unspliced in a polyadenylated transcript. Although many viral systems are known to translate proteins from mRNAs with retained introns, restriction mechanisms generally prevent export and translation of incompletely spliced mRNAs. Here, we provide evidence that the human nuclear poly(A)-binding protein, PABPN1, functions in such restrictions. Using a reporter construct in which nuclear export of an incompletely spliced mRNA is enhanced by a viral constitutive transport element (CTE), we show that PABPN1 depletion results in a significant increase in export and translation from the unspliced CTE-containing transcript. Unexpectedly, we find that inactivation of poly(A)-tail exosome targeting by depletion of PAXT components had no effect on export and translation of the unspliced reporter mRNA, suggesting a mechanism largely independent of nuclear RNA decay. Interestingly, a PABPN1 mutant selectively defective in stimulating poly(A) polymerase elongation strongly enhanced the expression of the unspliced, but not of intronless, reporter transcripts. Analysis of RNA-seq data also revealed that PABPN1 controls the expression of many human genes via intron retention. Notably, PABPN1-dependent intron retention events mostly affected 3'-terminal introns and were insensitive to PAXT and NEXT deficiencies. Our findings thus disclose a role for PABPN1 in restricting nuclear export of intron-retained transcripts and reinforce the interdependence between terminal intron splicing, 3' end processing, and polyadenylation.
Asunto(s)
Núcleo Celular , Empalme del ARN , Humanos , Intrones/genética , Transporte Activo de Núcleo Celular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , ARN Viral/genética , Expresión Génica , Proteína I de Unión a Poli(A)/genética , Proteína I de Unión a Poli(A)/metabolismoRESUMEN
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).
Asunto(s)
Nucleótidos , Tiouridina , ARN/genética , Rodaminas , Análisis de Secuencia de ARN/métodosRESUMEN
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.
Asunto(s)
Oxidorreductasas , ARN de Transferencia , Humanos , Oxidorreductasas/genética , ARN/química , ARN Mensajero/genética , ARN de Transferencia/química , ARN de Transferencia/genética , Uridina/químicaRESUMEN
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.
Asunto(s)
Segregación Cromosómica , Escherichia coli/genética , Meiosis , Procesamiento Postranscripcional del ARN , ARN Bacteriano/genética , ARN de Hongos/genética , ARN Mensajero/genética , Schizosaccharomyces/genética , Uridina/metabolismo , Cromosomas Bacterianos , Cromosomas Fúngicos , Cromosomas Humanos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolución Molecular , Células HCT116 , Humanos , Oxidación-Reducción , ARN Bacteriano/metabolismo , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Análisis de Secuencia de ARN , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
Transcription termination of protein-coding genes in eukaryotic cells usually relies on a tight coordination between the cleavage and polyadenylation of the pre-mRNA, and 5'-3' degradation of the downstream nascent transcript. Here we investigated the contribution of the essential fission yeast endonuclease Pac1, a homolog of human Drosha that cleaves hairpin RNA structures, in triggering polyadenylation-independent transcription termination. Using ChIP-sequencing in Pac1-deficient cells, we found that Pac1 triggers transcription termination at snRNA and snoRNA genes as well as at specific protein-coding genes. Notably, we found that Pac1-dependent premature termination occurred at two genes encoding conserved transmembrane transporters whose expression were strongly repressed by Pac1. Analysis by genome editing indicated that a stem-loop structure in the nascent transcript directs Pac1-mediated cleavage and that the regions upstream and downstream of the Pac1 cleavage site in the targeted mRNAs were stabilized by mutation of nuclear 3'-5' and 5'-3' exonucleases, respectively. Our findings unveil a premature transcription termination pathway that uncouples co-transcriptional RNA cleavage from polyadenylation, triggering rapid nuclear RNA degradation.
Asunto(s)
Endorribonucleasas/genética , ARN Nucleolar Pequeño/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Transcripción Genética , Humanos , Poliadenilación/genética , División del ARN/genética , ARN Polimerasa II/genética , ARN Mensajero/genética , Ribonucleasa III/genéticaRESUMEN
PDCD2 is an evolutionarily conserved protein with previously characterized homologs in Drosophila (zfrp8) and budding yeast (Tsr4). Although mammalian PDCD2 is essential for cell proliferation and embryonic development, the function of PDCD2 that underlies its fundamental cellular role has remained unclear. Here, we used quantitative proteomics approaches to define the protein-protein interaction network of human PDCD2. Our data revealed that PDCD2 specifically interacts with the 40S ribosomal protein uS5 (RPS2) and that the PDCD2-uS5 complex is assembled co-translationally. Loss of PDCD2 expression leads to defects in the synthesis of the small ribosomal subunit that phenocopy a uS5 deficiency. Notably, we show that PDCD2 is important for the accumulation of soluble uS5 protein as well as its incorporation into 40S ribosomal subunit. Our findings support that the essential molecular function of PDCD2 is to act as a dedicated ribosomal protein chaperone that recognizes uS5 co-translationally in the cytoplasm and accompanies uS5 to ribosome assembly sites in the nucleus. As most dedicated ribosomal protein chaperones have been identified in yeast, our study reveals that similar mechanisms exist in human cells to assist ribosomal proteins coordinate their folding, nuclear import and assembly in pre-ribosomal particles.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Chaperonas Moleculares/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Transporte Activo de Núcleo Celular/genética , Núcleo Celular/genética , Secuencia Conservada/genética , Células HeLa , Humanos , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Transcription by RNA polymerase II (RNAPII) is a dynamic process with frequent variations in the elongation rate. However, the physiological relevance of variations in RNAPII elongation kinetics has remained unclear. Here we show in yeast that a RNAPII mutant that reduces the transcription elongation rate causes widespread changes in alternative polyadenylation (APA). We unveil two mechanisms by which APA affects gene expression in the slow mutant: 3' UTR shortening and gene derepression by premature transcription termination of upstream interfering noncoding RNAs. Strikingly, the genes affected by these mechanisms are enriched for functions involved in phosphate uptake and purine synthesis, processes essential for maintenance of the intracellular nucleotide pool. As nucleotide concentration regulates transcription elongation, our findings argue that RNAPII is a sensor of nucleotide availability and that genes important for nucleotide pool maintenance have adopted regulatory mechanisms responsive to reduced rates of transcription elongation.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , ARN Polimerasa II/genética , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Activación Enzimática/efectos de los fármacos , Genes Fúngicos/genética , Mutación , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Fosfatos/farmacología , Poliadenilación , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Caenorhabditis elegans/genética , Biología Computacional/métodos , Empalme del ARN , ARN Lider Empalmado , Análisis de Secuencia de ARN/métodos , Trans-Empalme , Algoritmos , Animales , Genoma , Genómica/métodos , Operón , Precursores del ARN , ARN Mensajero/genética , Programas InformáticosRESUMEN
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.
Asunto(s)
ARN de Hongos/metabolismo , ARN Largo no Codificante/metabolismo , Schizosaccharomyces/fisiología , Ribonucleoproteína Nuclear Pequeña U2/genética , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/genética , Ensamble y Desensamble de Cromatina/genética , Nucleosomas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/genética , Adenosina Trifosfatasas/metabolismo , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Ensamble y Desensamble de Cromatina , Regulación Fúngica de la Expresión Génica , Histonas/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Factores de Transcripción/antagonistas & inhibidores , UbiquitinaciónRESUMEN
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.