RESUMEN
Ultraviolet (UV) irradiation and other genotoxic stresses induce bulky DNA lesions, which threaten genome stability and cell viability. Cells have evolved two main repair pathways to remove such lesions: global genome nucleotide excision repair (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER). The modes by which these subpathways recognize DNA lesions are distinct, but they converge onto the same downstream steps for DNA repair. Here, we first summarize the current understanding of these repair mechanisms, specifically focusing on the roles of stalled RNA polymerase II, Cockayne syndrome protein B (CSB), CSA and UV-stimulated scaffold protein A (UVSSA) in TC-NER. We also discuss the intriguing role of protein ubiquitylation in this process. Additionally, we highlight key aspects of the effect of UV irradiation on transcription and describe the role of signaling cascades in orchestrating this response. Finally, we describe the pathogenic mechanisms underlying xeroderma pigmentosum and Cockayne syndrome, the two main diseases linked to mutations in NER factors.
Asunto(s)
Síndrome de Cockayne , Humanos , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Transcripción Genética , Reparación del ADN , Daño del ADN , ADN/genética , ADN/metabolismo , Proteínas Portadoras/metabolismoRESUMEN
STK19 was proposed to be a cancer driver, and recent work by Yin et al. (2019) in Cell suggested that the frequently recurring STK19 D89N substitution represents a gain-of-function change, allowing increased phosphorylation of NRAS to enhance melanocyte transformation. Here we show that the STK19 gene has been incorrectly annotated, and that the expressed protein is 110 amino acids shorter than indicated by current databases. The "cancer driving" STK19 D89N substitution is thus outside the coding region. We also fail to detect evidence of the mutation affecting STK19 expression; instead, it is a UV signature mutation, found in the promoter of other genes as well. Furthermore, STK19 is exclusively nuclear and chromatin-associated, while no evidence for it being a kinase was found. The data in this Matters Arising article raise fundamental questions about the recently proposed role for STK19 in melanoma progression via a function as an NRAS kinase, suggested by Yin et al. (2019) in Cell. See also the response by Yin et al. (2020), published in this issue.
Asunto(s)
Melanoma , Recurrencia Local de Neoplasia , GTP Fosfohidrolasas/metabolismo , Genes ras , Humanos , Melanoma/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Proteínas Nucleares , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Transducción de SeñalRESUMEN
In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA-damage-response coordination. K1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery-persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells.
Asunto(s)
Daño del ADN , ARN Polimerasa II/metabolismo , Reparación del ADN , Células HEK293 , Humanos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Ubiquitinación , Rayos UltravioletaRESUMEN
Accurate regulation of mRNA termination is required for correct gene expression. Here, we describe a role for SCAF4 and SCAF8 as anti-terminators, suppressing the use of early, alternative polyadenylation (polyA) sites. The SCAF4/8 proteins bind the hyper-phosphorylated RNAPII C-terminal repeat domain (CTD) phosphorylated on both Ser2 and Ser5 and are detected at early, alternative polyA sites. Concomitant knockout of human SCAF4 and SCAF8 results in altered polyA selection and subsequent early termination, leading to expression of truncated mRNAs and proteins lacking functional domains and is cell lethal. While SCAF4 and SCAF8 work redundantly to suppress early mRNA termination, they also have independent, non-essential functions. SCAF8 is an RNAPII elongation factor, whereas SCAF4 is required for correct termination at canonical, distal transcription termination sites in the presence of SCAF8. Together, SCAF4 and SCAF8 coordinate the transition between elongation and termination, ensuring correct polyA site selection and RNAPII transcriptional termination in human cells.
Asunto(s)
ARN Polimerasa II/metabolismo , ARN Mensajero/biosíntesis , Proteínas de Unión al ARN/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Elongación de la Transcripción Genética , Terminación de la Transcripción Genética , Células HEK293 , Humanos , Poli A/genética , Poli A/metabolismo , Dominios Proteicos , ARN Polimerasa II/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Factores de Empalme Serina-Arginina/genéticaRESUMEN
The journey of RNA polymerase II (Pol II) as it transcribes a gene is anything but a smooth ride. Transcript elongation is discontinuous and can be perturbed by intrinsic regulatory barriers, such as promoter-proximal pausing, nucleosomes, RNA secondary structures and the underlying DNA sequence. More substantial blocking of Pol II translocation can be caused by other physiological circumstances and extrinsic obstacles, including other transcribing polymerases, the replication machinery and several types of DNA damage, such as bulky lesions and DNA double-strand breaks. Although numerous different obstacles cause Pol II stalling or arrest, the cell somehow distinguishes between them and invokes different mechanisms to resolve each roadblock. Resolution of Pol II blocking can be as straightforward as temporary backtracking and transcription elongation factor S-II (TFIIS)-dependent RNA cleavage, or as drastic as premature transcription termination or degradation of polyubiquitylated Pol II and its associated nascent RNA. In this Review, we discuss the current knowledge of how these different Pol II stalling contexts are distinguished by the cell, how they overlap with each other, how they are resolved and how, when unresolved, they can cause genome instability.
Asunto(s)
Nucleosomas , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Factores de Elongación Transcripcional/metabolismo , Animales , Humanos , Factores de Elongación Transcripcional/genéticaRESUMEN
The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal â¼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage.
Asunto(s)
Empalme Alternativo/efectos de la radiación , ADN Helicasas/genética , ARN no Traducido/genética , Transcripción Genética , Rayos Ultravioleta , Línea Celular , Exones , Humanos , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elongación de la Transcripción Genética/efectos de la radiación , Iniciación de la Transcripción Genética/efectos de la radiaciónRESUMEN
RNA polymerase II (RNAPII) transcription involves initiation from a promoter, transcriptional elongation through the gene, and termination in the terminator region. In bacteria, terminators often contain specific DNA elements provoking polymerase dissociation, but RNAPII transcription termination is thought to be driven entirely by protein co-factors. We used biochemical reconstitution, single-molecule studies, and genome-wide analysis in yeast to study RNAPII termination. Transcription into natural terminators by pure RNAPII results in spontaneous termination at specific sequences containing T-tracts. Single-molecule analysis indicates that termination involves pausing without backtracking. The "torpedo" Rat1-Rai1 exonuclease (XRN2 in humans) greatly stimulates spontaneous termination but is ineffectual on other paused RNAPIIs. By contrast, elongation factor Spt4-Spt5 (DSIF) suppresses termination. Genome-wide analysis further indicates that termination occurs by transcript cleavage at the poly(A) site exposing a new 5' RNA-end that allows Rat1-Rai1 loading, which then catches up with destabilized RNAPII at specific termination sites to end transcription.
Asunto(s)
ARN Polimerasa II , Proteínas de Saccharomyces cerevisiae , Humanos , ARN Polimerasa II/genética , ADN , Transcripción Genética , Exonucleasas , Factores de Elongación de Péptidos , Saccharomyces cerevisiae/genética , Proteínas de Unión al ARN , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The heat shock (HS) response involves rapid induction of HS genes, whereas transcriptional repression is established more slowly at most other genes. Previous data suggested that such repression results from inhibition of RNA polymerase II (RNAPII) pause release, but here, we show that HS strongly affects other phases of the transcription cycle. Intriguingly, while elongation rates increase upon HS, processivity markedly decreases, so that RNAPII frequently fails to reach the end of genes. Indeed, HS results in widespread premature transcript termination at cryptic, intronic polyadenylation (IPA) sites near gene 5'-ends, likely via inhibition of U1 telescripting. This results in dramatic reconfiguration of the human transcriptome with production of new, previously unannotated, short mRNAs that accumulate in the nucleus. Together, these results shed new light on the basic transcription mechanisms induced by growth at elevated temperature and show that a genome-wide shift toward usage of IPA sites can occur under physiological conditions.
Asunto(s)
Poliadenilación , Transcriptoma , Respuesta al Choque Térmico/genética , Humanos , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/genéticaRESUMEN
RECQL5 is the sole member of the RECQ family of helicases associated with RNA polymerase II (RNAPII). We now show that RECQL5 is a general elongation factor that is important for preserving genome stability during transcription. Depletion or overexpression of RECQL5 results in corresponding shifts in the genome-wide RNAPII density profile. Elongation is particularly affected, with RECQL5 depletion causing a striking increase in the average rate, concurrent with increased stalling, pausing, arrest, and/or backtracking (transcription stress). RECQL5 therefore controls the movement of RNAPII across genes. Loss of RECQL5 also results in the loss or gain of genomic regions, with the breakpoints of lost regions located in genes and common fragile sites. The chromosomal breakpoints overlap with areas of elevated transcription stress, suggesting that RECQL5 suppresses such stress and its detrimental effects, and thereby prevents genome instability in the transcribed region of genes.
Asunto(s)
Inestabilidad Genómica , RecQ Helicasas/metabolismo , Elongación de la Transcripción Genética , Transcripción Genética , Genoma Humano , Células HEK293 , Humanos , ARN Polimerasa II/metabolismoRESUMEN
The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway senses cytosolic DNA and induces interferon-stimulated genes (ISGs) to activate the innate immune system. Here, we report the unexpected discovery that cGAS also senses dysfunctional protein production. Purified ribosomes interact directly with cGAS and stimulate its DNA-dependent activity in vitro. Disruption of the ribosome-associated protein quality control (RQC) pathway, which detects and resolves ribosome collision during translation, results in cGAS-dependent ISG expression and causes re-localization of cGAS from the nucleus to the cytosol. Indeed, cGAS preferentially binds collided ribosomes in vitro, and orthogonal perturbations that result in elevated levels of collided ribosomes and RQC activation cause sub-cellular re-localization of cGAS and ribosome binding in vivo as well. Thus, translation stress potently increases DNA-dependent cGAS activation. These findings have implications for the inflammatory response to viral infection and tumorigenesis, both of which substantially reprogram cellular protein synthesis.
Asunto(s)
Núcleo Celular , Nucleotidiltransferasas , Biosíntesis de Proteínas , Ribosomas , Transducción de Señal , Estrés Fisiológico , Transporte Activo de Núcleo Celular , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Ribosomas/química , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Human synovial sarcoma is caused by a chromosome translocation, which fuses DNA encoding SSX to that encoding the SS18 protein. Kadoch and Crabtree now show that the resulting cellular transformation stems from disruption of the normal architecture and function of the human SWI/SNF (BAF) complex.
RESUMEN
DNA damage triggers polyubiquitylation and degradation of the largest subunit of RNA polymerase II (RNAPII), a "mechanism of last resort" employed during transcription stress. In yeast, this process is dependent on Def1 through a previously unresolved mechanism. Here, we report that Def1 becomes activated through ubiquitylation- and proteasome-dependent processing. Def1 processing results in the removal of a domain promoting cytoplasmic localization, resulting in nuclear accumulation of the clipped protein. Nuclear Def1 then binds RNAPII, utilizing a ubiquitin-binding domain to recruit the Elongin-Cullin E3 ligase complex via a ubiquitin-homology domain in the Ela1 protein. This facilitates polyubiquitylation of Rpb1, triggering its proteasome-mediated degradation. Together, these results outline the multistep mechanism of Rpb1 polyubiquitylation triggered by transcription stress and uncover the key role played by Def1 as a facilitator of Elongin-Cullin ubiquitin ligase function.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Transcripción Genética , Secuencia de Aminoácidos , Proteínas Cromosómicas no Histona/química , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Estructura Terciaria de Proteína , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Alineación de Secuencia , Estrés Fisiológico , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
Translating ribosomes that slow excessively incur collisions with trailing ribosomes. Persistent collisions are detected by ZNF598, a ubiquitin ligase that ubiquitinates sites on the ribosomal 40S subunit to initiate pathways of mRNA and protein quality control. The collided ribosome complex must be disassembled to initiate downstream quality control, but the mechanistic basis of disassembly is unclear. Here, we reconstitute the disassembly of a collided polysome in a mammalian cell-free system. The widely conserved ASC-1 complex (ASCC) containing the ASCC3 helicase disassembles the leading ribosome in an ATP-dependent reaction. Disassembly, but not ribosome association, requires 40S ubiquitination by ZNF598, but not GTP-dependent factors, including the Pelo-Hbs1L ribosome rescue complex. Trailing ribosomes can elongate once the roadblock has been removed and only become targets if they subsequently stall and incur collisions. These findings define the specific role of ASCC during ribosome-associated quality control and identify the molecular target of its activity.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/metabolismo , Complejos Multiproteicos/metabolismo , Biosíntesis de Proteínas , Ribosomas/metabolismo , Sistema de Transporte de Aminoácidos y+/genética , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Sistema Libre de Células , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Polirribosomas/genética , Polirribosomas/metabolismo , Conejos , Subunidades Ribosómicas/genética , Subunidades Ribosómicas/metabolismo , Ribosomas/genética , UbiquitinaciónRESUMEN
The Ddi1/DDI2 proteins are ubiquitin shuttling factors, implicated in a variety of cellular functions. In addition to ubiquitin-binding and ubiquitin-like domains, they contain a conserved region with similarity to retroviral proteases, but whether and how DDI2 functions as a protease has remained unknown. Here, we show that DDI2 knockout cells are sensitive to proteasome inhibition and accumulate high-molecular weight, ubiquitylated proteins that are poorly degraded by the proteasome. These proteins are targets for the protease activity of purified DDI2. No evidence for DDI2 acting as a de-ubiquitylating enzyme was uncovered, which could suggest that it cleaves the ubiquitylated protein itself. In support of this idea, cleavage of transcription factor NRF1 is known to require DDI2 activity in vivo. We show that DDI2 is indeed capable of cleaving NRF1 in vitro but only when NRF1 protein is highly poly-ubiquitylated. Together, these data suggest that DDI2 is a ubiquitin-directed endoprotease.
Asunto(s)
Proteasas de Ácido Aspártico/metabolismo , Factor Nuclear 1 de Respiración/metabolismo , Ubiquitina/metabolismo , Proteasas de Ácido Aspártico/genética , Sitios de Unión , Sistemas CRISPR-Cas , Línea Celular , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Biosíntesis de Proteínas , ProteolisisRESUMEN
In this issue of Molecular Cell, Nicholls et al. (2019) show that the oligoribonuclease REXO2 degrades mitochondrial RNA dinucleotides to prevent RNA-primed transcription at non-canonical sites in the mitochondrial genome, shedding new light on the importance of complete RNA degradation for transcriptional integrity.
Asunto(s)
Mitocondrias , Transcripción Genética , Animales , Mitofagia , Regiones Promotoras Genéticas , ARNRESUMEN
Although correlations between RNA polymerase II (RNAPII) transcription stress, R-loops, and genome instability have been established, the mechanisms underlying these connections remain poorly understood. Here, we used a mutant version of the transcription elongation factor TFIIS (TFIISmut), aiming to specifically induce increased levels of RNAPII pausing, arrest, and/or backtracking in human cells. Indeed, TFIISmut expression results in slower elongation rates, relative depletion of polymerases from the end of genes, and increased levels of stopped RNAPII; it affects mRNA splicing and termination as well. Remarkably, TFIISmut expression also dramatically increases R-loops, which may form at the anterior end of backtracked RNAPII and trigger genome instability, including DNA strand breaks. These results shed light on the relationship between transcription stress and R-loops and suggest that different classes of R-loops may exist, potentially with distinct consequences for genome stability.
Asunto(s)
Inestabilidad Genómica , Estructuras R-Loop , ARN Mensajero/genética , Estrés Fisiológico , Transcripción Genética , Factores de Elongación Transcripcional/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Mutación , ARN Polimerasa II/metabolismo , Empalme del ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , Relación Estructura-Actividad , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/genéticaRESUMEN
Until recently, it was generally assumed that essentially all regulation of transcription takes place via regions adjacent to the coding region of a gene--namely promoters and enhancers--and that, after recruitment to the promoter, the polymerase simply behaves like a machine, quickly "reading the gene." However, over the past decade a revolution in this thinking has occurred, culminating in the idea that transcript elongation is extremely complex and highly regulated and, moreover, that this process significantly affects both the organization and integrity of the genome. This review addresses basic aspects of transcript elongation by RNA polymerase II (RNAPII) and how it relates to other DNA-related processes.
Asunto(s)
Regulación de la Expresión Génica , ARN Polimerasa II/metabolismo , Transcripción Genética , Animales , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , HumanosRESUMEN
RECQL5 is a member of the conserved RecQ family of DNA helicases involved in the maintenance of genome stability that is specifically found in higher eukaryotes and associates with the elongating RNA polymerase II. To expand our understanding of its function we expressed human RECQL5 in the yeast Saccharomyces cerevisiae, which does not have a RECQL5 ortholog. We found that RECQL5 expression leads to cell growth inhibition, increased genotoxic sensitivity and transcription-associated hyperrecombination. Chromatin immunoprecipitation and transcriptomic analysis of yeast cells expressing human RECQL5 shows that this is recruited to transcribed genes and although it causes only a weak impact on gene expression, in particular at G + C-rich genes, it leads to a transcription termination defect detected as readthrough transcription. The data indicate that the interaction between RNAPII and RECQL5 is conserved from yeast to humans. Unexpectedly, however, the RECQL5-ID mutant, previously shown to have reduced the association with RNAPII in vitro, associates with the transcribing polymerase in cells. As a result, expression of RECQL5-ID leads to similar although weaker phenotypes than wild-type RECQL5 that could be transcription-mediated. Altogether, the data suggests that RECQL5 has the intrinsic ability to function in transcription-dependent and independent genome dynamics in S. cerevisiae.
Asunto(s)
Inestabilidad Genómica , RecQ Helicasas , Saccharomyces cerevisiae , Transcripción Genética , Saccharomyces cerevisiae/genética , Inestabilidad Genómica/genética , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Humanos , Transcripción Genética/genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismoRESUMEN
Genome instability is a recurring feature of tumorigenesis. Mutation in MLL2, encoding a histone methyltransferase, is a driver in numerous different cancer types, but the mechanism is unclear. Here, we present evidence that MLL2 mutation results in genome instability. Mouse cells in which MLL2 gene deletion can be induced display elevated levels of sister chromatid exchange, gross chromosomal aberrations, 53BP1 foci, and micronuclei. Human MLL2 knockout cells are characterized by genome instability as well. Interestingly, MLL2 interacts with RNA polymerase II (RNAPII) and RECQL5, and, although MLL2 mutated cells have normal overall H3K4me levels in genes, nucleosomes in the immediate vicinity of RNAPII are hypomethylated. Importantly, MLL2 mutated cells display signs of substantial transcription stress, and the most affected genes overlap with early replicating fragile sites, show elevated levels of γH2AX, and suffer frequent mutation. The requirement for MLL2 in the maintenance of genome stability in genes helps explain its widespread role in cancer and points to transcription stress as a strong driver in tumorigenesis.