RESUMEN

DNA-protein crosslinks (DPCs) challenge faithful DNA replication and smooth passage of genomic information. Our study unveils the cullin E3 ubiquitin ligase Rtt101 as a DPC repair factor. Genetic analyses demonstrate that Rtt101 is essential for resistance to a wide range of DPC types including topoisomerase 1 crosslinks, in the same pathway as the ubiquitin-dependent aspartic protease Ddi1. Using an in vivo inducible Top1-mimicking DPC system, we reveal the significant impact of Rtt101 ubiquitination on DPC removal across different cell cycle phases. High-throughput methods coupled with next-generation sequencing specifically highlight the association of Rtt101 with replisomes as well as colocalization with DPCs. Our findings establish Rtt101 as a main contributor to DPC repair throughout the yeast cell cycle.

Asunto(s)

Ciclo Celular , Proteínas Cullin , Reparación del ADN , Proteínas de Saccharomyces cerevisiae , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ciclo Celular/genética , Saccharomyces cerevisiae , Aductos de ADN/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , Transporte de Proteínas/genética , Ubiquitinación/genética , Replicación del ADN/genética , Complejos Multienzimáticos/metabolismo

RESUMEN

Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. A negative role for antisense transcription at the PHO5 gene locus was demonstrated by leveraging the level of overlapping antisense transcription through specific mutant backgrounds, expression from a strong promoter in cis, and use of the CRISPRi system. Furthermore, we showed that enhanced elongation of PHO5 antisense leads to a more repressive chromatin conformation at the PHO5 gene promoter, which is more slowly remodeled upon gene induction. The negative effect of antisense transcription on PHO5 gene transcription is mitigated upon inactivation of the histone deacetylase Rpd3, showing that PHO5 antisense RNA acts via histone deacetylation. This regulatory pathway leads to Rpd3-dependent decreased recruitment of the RSC chromatin remodeling complex to the PHO5 gene promoter upon induction of antisense transcription. Overall, the data in this work reveal an additional level in the complex regulatory mechanism of PHO5 gene expression by showing antisense transcription-mediated repression at the level of promoter chromatin structure remodeling.

Asunto(s)

Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Histonas/genética , Fosfatasa Ácida/genética , Fosfatasa Ácida/metabolismo , Cromatina/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , ARN sin Sentido/genética , Transcripción Genética , Regulación Fúngica de la Expresión Génica

RESUMEN

Eukaryotic genomes are pervasively transcribed by RNA polymerase II (RNAPII), and transcription of long non-coding RNAs often overlaps with coding gene promoters. This might lead to coding gene repression in a process named Transcription Interference (TI). In Saccharomyces cerevisiae, TI is mainly driven by antisense non-coding transcription and occurs through re-shaping of promoter Nucleosome-Depleted Regions (NDRs). In this study, we developed a genetic screen to identify new players involved in Antisense-Mediated Transcription Interference (AMTI). Among the candidates, we found the HIR histone chaperone complex known to be involved in de novo histone deposition. Using genome-wide approaches, we reveal that HIR-dependent histone deposition represses the promoters of SAGA-dependent genes via antisense non-coding transcription. However, while antisense transcription is enriched at promoters of SAGA-dependent genes, this feature is not sufficient to define the mode of gene regulation. We further show that the balance between HIR-dependent nucleosome incorporation and transcription factor binding at promoters directs transcription into a SAGA- or TFIID-dependent regulation. This study sheds light on a new connection between antisense non-coding transcription and the nature of coding transcription initiation.

Asunto(s)

Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Regulación Fúngica de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción Genética

RESUMEN

Transcription in eukaryotes correlates with major chromatin changes, including the replacement of old nucleosomal histones by new histones at the promoters of genes. The role of these histone exchange events in transcription remains unclear. In particular, the causal relationship between histone exchange and activator binding, preinitiation complex (PIC) assembly, and/or subsequent transcription remains unclear. Here, we provide evidence that histone exchange at gene promoters is not simply a consequence of PIC assembly or transcription but instead is mediated by activators. We further show that not all activators up-regulate gene expression by inducing histone turnover. Thus, histone exchange does not simply correlate with transcriptional activity, but instead reflects the mode of action of the activator. Last, we show that histone turnover is not only associated with activator function but also plays a role in transcriptional repression at the histone loci.

Asunto(s)

Ensamble y Desensamble de Cromatina , Histonas , Cromatina/genética , Inmunoprecipitación de Cromatina , Histonas/genética , Histonas/metabolismo , Regiones Promotoras Genéticas , Transcripción Genética

RESUMEN

Eukaryotic genomes are almost entirely transcribed by RNA polymerase II. Consequently, the transcription of long noncoding RNAs often overlaps with coding gene promoters, triggering potential gene repression through a poorly characterized mechanism of transcription interference. Here, we propose a comprehensive model of chromatin-based transcription interference in Saccharomyces cerevisiae (S. cerevisiae). By using a noncoding transcription-inducible strain, we analyze the relationship between antisense elongation and coding sense repression, nucleosome occupancy, and transcription-associated histone modifications using near-base pair resolution techniques. We show that antisense noncoding transcription leads to the deacetylation of a subpopulation of -1/+1 nucleosomes associated with increased H3K36me3. Reduced acetylation results in the decreased binding of the RSC chromatin remodeler at -1/+1 nucleosomes and subsequent sliding into the nucleosome-depleted region hindering pre-initiation complex association. Finally, we extend our model by showing that natural antisense noncoding transcription significantly represses ∼20% of S. cerevisiae genes through this chromatin-based transcription interference mechanism.

Asunto(s)

Cromatina/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Histonas/metabolismo , Nucleosomas/metabolismo , Factores de Transcripción/genética , Ensamble y Desensamble de Cromatina/genética , Regiones Promotoras Genéticas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genética

RESUMEN

RNA polymerase II (RNAP II) non-coding transcription is now known to cover almost the entire eukaryotic genome, a phenomenon referred to as pervasive transcription. As a consequence, regions previously thought to be non-transcribed are subject to the passage of RNAP II and its associated proteins for histone modification. This is the case for the nucleosome-depleted regions (NDRs), which provide key sites of entry into the chromatin for proteins required for the initiation of coding gene transcription and DNA replication. In this review, recent data on the effects of pervasive transcription through NDRs are summarized and a model is proposed to explain how RNAP II-driven transcription is able to modify the nucleosomes flanking the NDRs, leading to nucleosome repositioning and NDR closure. Even though much of the mechanistic detail underlying these events remains to be elucidated, such a model provides a basis to explain how non-coding transcription through NDRs can regulate the initiation of coding gene expression and DNA replication.

Asunto(s)

Cromatina/genética , Replicación del ADN/genética , Expresión Génica/genética , Nucleosomas/genética , ARN no Traducido/genética , Transcripción Genética/genética , Humanos , ARN Polimerasa II/genética

RESUMEN

In eukaryotic organisms, replication initiation follows a temporal program. Among the parameters that regulate this program in Saccharomyces cerevisiae, chromatin structure has been at the center of attention without considering the contribution of transcription. Here, we revisit the replication initiation program in the light of widespread genomic noncoding transcription. We find that noncoding RNA transcription termination in the vicinity of autonomously replicating sequences (ARSs) shields replication initiation from transcriptional readthrough. Consistently, high natural nascent transcription correlates with low ARS efficiency and late replication timing. High readthrough transcription is also linked to increased nucleosome occupancy and high levels of H3K36me3. Moreover, forcing ARS readthrough transcription promotes these chromatin features. Finally, replication initiation defects induced by increased transcriptional readthrough are partially rescued in the absence of H3K36 methylation. Altogether, these observations indicate that natural noncoding transcription into ARSs influences replication initiation through chromatin regulation.

Asunto(s)

Cromatina/genética , Replicación del ADN , Regulación de la Expresión Génica , ARN no Traducido , Acetilación , Regulación Fúngica de la Expresión Génica , Histonas/metabolismo , Nucleosomas/metabolismo , ARN Mensajero/genética , Origen de Réplica , Saccharomyces cerevisiae/genética , Transcripción Genética

RESUMEN

The model for telomere shortening at each replication cycle is currently incomplete, and the exact contribution of the telomeric 3' overhang to the shortening rate remains unclear. Here, we demonstrate key steps of the mechanism of telomere replication in Saccharomyces cerevisiae. By following the dynamics of telomeres during replication at near-nucleotide resolution, we find that the leading-strand synthesis generates blunt-end intermediates before being 5'-resected and filled in. Importantly, the shortening rate is set by positioning the last Okazaki fragments at the very ends of the chromosome. Thus, telomeres shorten in direct proportion to the 3' overhang lengths of 5-10 nucleotides that are present in parental templates. Furthermore, the telomeric protein Cdc13 coordinates leading- and lagging-strand syntheses. Taken together, our data unravel a precise choreography of telomere replication elucidating the DNA end-replication problem and provide a framework to understand the control of the cell proliferation potential.

Asunto(s)

Cromosomas Fúngicos , Replicación del ADN , ADN de Cadena Simple , Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/genética , Telómero/química , Proliferación Celular , ADN/química , ADN/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homeostasis del Telómero , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo

RESUMEN

In the absence of telomerase, telomeres progressively shorten with every round of DNA replication, leading to replicative senescence. In telomerase-deficient Saccharomyces cerevisiae, the shortest telomere triggers the onset of senescence by activating the DNA damage checkpoint and recruiting homologous recombination (HR) factors. Yet, the molecular structures that trigger this checkpoint and the mechanisms of repair have remained elusive. By tracking individual telomeres, we show that telomeres are subjected to different pathways depending on their length. We first demonstrate a progressive accumulation of subtelomeric single-stranded DNA (ssDNA) through 5'-3' resection as telomeres shorten. Thus, exposure of subtelomeric ssDNA could be the signal for cell cycle arrest in senescence. Strikingly, early after loss of telomerase, HR counteracts subtelomeric ssDNA accumulation rather than elongates telomeres. We then asked whether replication repair pathways contribute to this mechanism. We uncovered that Rad5, a DNA helicase/Ubiquitin ligase of the error-free branch of the DNA damage tolerance (DDT) pathway, associates with native telomeres and cooperates with HR in senescent cells. We propose that DDT acts in a length-independent manner, whereas an HR-based repair using the sister chromatid as a template buffers precocious 5'-3' resection at the shortest telomeres.

Asunto(s)

Reparación del ADN por Recombinación , Acortamiento del Telómero , ADN Helicasas/análisis , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Fase G2/genética , RecQ Helicasas/metabolismo , Fase S/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/metabolismo , Telomerasa/genética , Telómero/química , Homeostasis del Telómero

RESUMEN

Maturation of the 40S ribosomal subunit precursors in mammals mobilizes several non-ribosomal proteins, including the atypical protein kinase RioK2. Here, we have investigated the involvement of another member of the RIO kinase family, RioK3, in human ribosome biogenesis. RioK3 is a cytoplasmic protein that does not seem to shuttle between nucleus and cytoplasm via a Crm1-dependent mechanism as does RioK2 and which sediments with cytoplasmic 40S ribosomal particles in a sucrose gradient. When the small ribosomal subunit biogenesis is impaired by depletion of either rpS15, rpS19 or RioK2, a concomitant decrease in the amount of RioK3 is observed. Surprisingly, we observed a dramatic and specific increase in the levels of RioK3 when the biogenesis of the large ribosomal subunit is impaired. A fraction of RioK3 is associated with the non ribosomal pre-40S particle components hLtv1 and hEnp1 as well as with the 18S-E pre-rRNA indicating that it belongs to a bona fide cytoplasmic pre-40S particle. Finally, RioK3 depletion leads to an increase in the levels of the 21S rRNA precursor in the 18S rRNA production pathway. Altogether, our results strongly suggest that RioK3 is a novel cytoplasmic component of pre-40S pre-ribosomal particle(s) in human cells, required for normal processing of the 21S pre-rRNA.

Asunto(s)

Citoplasma/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Precursores del ARN/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Células HeLa , Humanos , Datos de Secuencia Molecular , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , ARN Ribosómico 18S/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Alineación de Secuencia

RESUMEN

It is generally assumed that, in Saccharomyces cerevisiae, immature 40S ribosomal subunits are not competent for translation initiation. Here, we show by different approaches that, in wild-type conditions, a portion of pre-40S particles (pre-SSU) associate with translating ribosomal complexes. When cytoplasmic 20S pre-rRNA processing is impaired, as in Rio1p- or Nob1p-depleted cells, a large part of pre-SSUs is associated with translating ribosomes complexes. Loading of pre-40S particles onto mRNAs presumably uses the canonical pathway as translation-initiation factors interact with 20S pre-rRNA. However, translation initiation is not required for 40S ribosomal subunit maturation. We also provide evidence suggesting that cytoplasmic 20S pre-rRNAs that associate with translating complexes are turned over by the no go decay (NGD) pathway, a process known to degrade mRNAs on which ribosomes are stalled. We propose that the cytoplasmic fate of 20S pre-rRNA is determined by the balance between pre-SSU processing kinetics and sensing of ribosome-like particles loaded onto mRNAs by the NGD machinery, which acts as an ultimate ribosome quality check point.

Asunto(s)

Iniciación de la Cadena Peptídica Traduccional , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , ADN de Hongos/genética , Factor 1 Eucariótico de Iniciación/genética , Factor 1 Eucariótico de Iniciación/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Cinética , Modelos Biológicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Polirribosomas/genética , Polirribosomas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN de Hongos/genética , ARN de Hongos/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/genética , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo