RESUMEN
Termination of RNA polymerase II (RNAPII) transcription in metazoans relies largely on the cleavage and polyadenylation (CPA) and integrator (INT) complexes originally found to act at the ends of protein-coding and small nuclear RNA (snRNA) genes, respectively. Here, we monitor CPA- and INT-dependent termination activities genome-wide, including at thousands of previously unannotated transcription units (TUs), producing unstable RNA. We verify the global activity of CPA occurring at pA sites indiscriminately of their positioning relative to the TU promoter. We also identify a global activity of INT, which is largely sequence-independent and restricted to a ~3-kb promoter-proximal region. Our analyses suggest two functions of genome-wide INT activity: it dampens transcriptional output from weak promoters, and it provides quality control of RNAPII complexes that are unfavorably configured for transcriptional elongation. We suggest that the function of INT in stable snRNA production is an exception from its general cellular role, the attenuation of non-productive transcription.
Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Proteínas de Unión al ADN/metabolismo , ARN Polimerasa II/metabolismo , ARN Nuclear Pequeño/biosíntesis , Terminación de la Transcripción Genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Poliadenilación , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , ARN Nuclear Pequeño/genéticaRESUMEN
Spt5 is an essential and conserved factor that functions in transcription and co-transcriptional processes. However, many aspects of the requirement for Spt5 in transcription are poorly understood. We have analyzed the consequences of Spt5 depletion in Schizosaccharomyces pombe using four genome-wide approaches. Our results demonstrate that Spt5 is crucial for a normal rate of RNA synthesis and distribution of RNAPII over transcription units. In the absence of Spt5, RNAPII localization changes dramatically, with reduced levels and a relative accumulation over the first â¼500 bp, suggesting that Spt5 is required for transcription past a barrier. Spt5 depletion also results in widespread antisense transcription initiating within this barrier region. Deletions of this region alter the distribution of RNAPII on the sense strand, suggesting that the barrier observed after Spt5 depletion is normally a site at which Spt5 stimulates elongation. Our results reveal a global requirement for Spt5 in transcription elongation.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , ARN sin Sentido/biosíntesis , ARN de Hongos/biosíntesis , ARN Mensajero/biosíntesis , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Elongación de la Transcripción Genética , Factores de Elongación Transcripcional/metabolismo , Proteínas Cromosómicas no Histona/genética , Biología Computacional , Bases de Datos Genéticas , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Genotipo , Mutación , Fenotipo , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Empalme del ARN , ARN sin Sentido/genética , ARN de Hongos/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Factores de Tiempo , Factores de Elongación Transcripcional/genéticaRESUMEN
To monitor transcriptional regulation in human cells, rapid changes in enhancer and promoter activity must be captured with high sensitivity and temporal resolution. Here, we show that the recently established protocol TT-seq ("transient transcriptome sequencing") can monitor rapid changes in transcription from enhancers and promoters during the immediate response of T cells to ionomycin and phorbol 12-myristate 13-acetate (PMA). TT-seq maps eRNAs and mRNAs every 5 min after T-cell stimulation with high sensitivity and identifies many new primary response genes. TT-seq reveals that the synthesis of 1,601 eRNAs and 650 mRNAs changes significantly within only 15 min after stimulation, when standard RNA-seq does not detect differentially expressed genes. Transcription of enhancers that are primed for activation by nucleosome depletion can occur immediately and simultaneously with transcription of target gene promoters. Our results indicate that enhancer transcription is a good proxy for enhancer regulatory activity in target gene activation, and establish TT-seq as a tool for monitoring the dynamics of enhancer landscapes and transcription programs during cellular responses and differentiation.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Ionomicina/farmacología , Análisis de Secuencia de ARN/métodos , Linfocitos T/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Emparejamiento Base , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Jurkat , ARN/análisis , Transcripción Genética/efectos de los fármacos , Activación TranscripcionalRESUMEN
To decrypt the regulatory code of the genome, sequence elements must be defined that determine the kinetics of RNA metabolism and thus gene expression. Here, we attempt such decryption in an eukaryotic model organism, the fission yeast S. pombe. We first derive an improved genome annotation that redefines borders of 36% of expressed mRNAs and adds 487 non-coding RNAs (ncRNAs). We then combine RNA labeling in vivo with mathematical modeling to obtain rates of RNA synthesis and degradation for 5,484 expressed RNAs and splicing rates for 4,958 introns. We identify functional sequence elements in DNA and RNA that control RNA metabolic rates and quantify the contributions of individual nucleotides to RNA synthesis, splicing, and degradation. Our approach reveals distinct kinetics of mRNA and ncRNA metabolism, separates antisense regulation by transcription interference from RNA interference, and provides a general tool for studying the regulatory code of genomes.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Genoma Fúngico , ARN de Hongos/genética , ARN Mensajero/genética , Schizosaccharomyces/genética , Intrones , Interferencia de ARN , Empalme del ARN , ARN sin Sentido/genética , Análisis de Secuencia de ARN , Transcripción GenéticaRESUMEN
During the cell cycle, the levels of hundreds of mRNAs change in a periodic manner, but how this is achieved by alterations in the rates of mRNA synthesis and degradation has not been studied systematically. Here, we used metabolic RNA labeling and comparative dynamic transcriptome analysis (cDTA) to derive mRNA synthesis and degradation rates every 5 min during three cell cycle periods of the yeast Saccharomyces cerevisiae. A novel statistical model identified 479 genes that show periodic changes in mRNA synthesis and generally also periodic changes in their mRNA degradation rates. Peaks of mRNA degradation generally follow peaks of mRNA synthesis, resulting in sharp and high peaks of mRNA levels at defined times during the cell cycle. Whereas the timing of mRNA synthesis is set by upstream DNA motifs and their associated transcription factors (TFs), the synthesis rate of a periodically expressed gene is apparently set by its core promoter.
Asunto(s)
Perfilación de la Expresión Génica , Genes cdc , Estabilidad del ARN/genética , ARN Mensajero/biosíntesis , Ciclo Celular/genética , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Enhancer activity drives cell differentiation and cell fate determination, but it remains unclear how enhancers cooperate during these processes. Here we investigate enhancer cooperation during transdifferentiation of human leukemia B-cells to macrophages. Putative enhancers are established by binding of the pioneer factor C/EBPα followed by chromatin opening and enhancer RNA (eRNA) synthesis from H3K4-monomethylated regions. Using eRNA synthesis as a proxy for enhancer activity, we find that most putative enhancers cooperate in an additive way to regulate transcription of assigned target genes. However, transcription from 136 target genes depends exponentially on the summed activity of its putative paired enhancers, indicating that these enhancers cooperate synergistically. The target genes are cell type-specific, suggesting that enhancer synergy can contribute to cell fate determination. Enhancer synergy appears to depend on cell type-specific transcription factors, and such interacting enhancers are not predicted from occupancy or accessibility data that are used to detect superenhancers.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Diferenciación Celular/genética , Histonas/metabolismo , ARN/metabolismo , Transcripción Genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Cromatina/metabolismo , Elementos de Facilitación Genéticos , Humanos , Regiones Promotoras Genéticas , Células THP-1RESUMEN
Pervasive transcription of the genome produces both stable and transient RNAs. We developed transient transcriptome sequencing (TT-seq), a protocol that uniformly maps the entire range of RNA-producing units and estimates rates of RNA synthesis and degradation. Application of TT-seq to human K562 cells recovers stable messenger RNAs and long intergenic noncoding RNAs and additionally maps transient enhancer, antisense, and promoter-associated RNAs. TT-seq analysis shows that enhancer RNAs are short-lived and lack U1 motifs and secondary structure. TT-seq also maps transient RNA downstream of polyadenylation sites and uncovers sites of transcription termination; we found, on average, four transcription termination sites, distributed in a window with a median width of ~3300 base pairs. Termination sites coincide with a DNA motif associated with pausing of RNA polymerase before its release from the genome.