In response to transcription-blocking DNA lesions such as those generated by UV irradiation, cells activate a multipronged DNA damage response. This response encompasses repair of the lesions that ...stall RNA polymerase (RNAP) but also a poorly understood, genome-wide shutdown of transcription, even of genes that are not damaged. Over the past few years, a number of new results have shed light on this intriguing DNA damage response at the structural, biochemical, cell biological, and systems biology level. In this review we summarize the most important findings.
Biochemical and genetic studies indicate crosstalk between TC-NER factors and factors regulating RNAPII backtracking and elongation.
TC-NER is controlled by an unexpectedly complex layer of post-translational regulation targeting repair factors and RNAPII itself.
Dramatic changes to the transcriptional program in response to UV irradiation have been revealed by high-throughput genome-wide and time-resolved measurements.
Chromatin remodelers, splicing factors, noncoding RNAs, and RNA modifications provide a multifaceted transcriptional response to UV irradiation.
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.
Antisense noncoding transcripts, genes-within-genes, and convergent gene pairs are prevalent among eukaryotes. The existence of such transcription units raises the question of what happens when RNA ...polymerase II (RNAPII) molecules collide head-to-head. Here we use a combination of biochemical and genetic approaches in yeast to show that polymerases transcribing opposite DNA strands cannot bypass each other. RNAPII stops but does not dissociate upon head-to-head collision in vitro, suggesting that opposing polymerases represent insurmountable obstacles for each other. Head-to-head collision in vivo also results in RNAPII stopping, and removal of collided RNAPII from the DNA template can be achieved via ubiquitylation-directed proteolysis. Indeed, in cells lacking efficient RNAPII polyubiquitylation, the half-life of collided polymerases increases, so that they can be detected between convergent genes. These results provide insight into fundamental mechanisms of gene traffic control and point to an unexplored effect of antisense transcription on gene regulation via polymerase collision.
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► Convergently transcribing RNAPIIs cannot transcribe past one another in vivo ► In vitro, RNAPII stops when the front edges of the colliding proteins touch ► Collided polymerases remain stably associated with the template ► Collided RNAPII accumulates between convergent genes in elc1Δ strains
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.
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•DDI2 KO cells accumulate highly ubiquitylated proteins that are slowly degraded•DDI2 KO cells are hypersensitive to pharmacological proteasome inhibition•Purified DDI2 protein can directly cleave the highly ubiquitylated proteins in vitro•DDI2 site-specifically cuts NRF1 in vitro but only if NRF1 is poly-ubiquitylated
Dirac-Svejstrup et al. show that DDI2 knockout cells accumulate highly ubiquitylated proteins that are difficult to degrade and that these cells are hypersensitive to pharmacological proteasome inhibition. The DDI2 protein can directly cleave the highly ubiquitylated proteins in vitro and does this site specifically in the ubiquitylated transcription factor NRF1.
Lesions in genes that result in RNA polymerase II (RNAPII) stalling or arrest are particularly toxic as they are a focal point of genome instability and potently block further transcription of the ...affected gene. Thus, cells have evolved the transcription-coupled nucleotide excision repair (TC-NER) pathway to identify damage-stalled RNAPIIs, so that the lesion can be rapidly repaired and transcription can continue. However, despite the identification of several factors required for TC-NER, how RNAPII is remodelled, modified, removed, or whether this is even necessary for repair remains enigmatic, and theories are intensely contested. Recent studies have further detailed the cellular response to UV-induced ubiquitylation and degradation of RNAPII and its consequences for transcription and repair. These advances make it pertinent to revisit the TC-NER process in general and with specific discussion of the fate of RNAPII stalled at DNA lesions.
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.
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•Inducing transcription stress via TFIISmut results in reduced nascent RNA synthesis•TFIISmut affects expression of long genes and mRNA splicing•TFIISmut expression leads to increased R-loop formation and genomic instability•R-loops may form both anterior and posterior of RNAPII
Inducing transcription stress via expression of a TFIIS mutant results in increased R-loop formation. The R-loops may be formed anterior to the backtracking RNA polymerase II and result in increased genomic instability.
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.
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•ASC-1 Complex (ASCC) disassembles collided ribosomes ubiquitinated by ZNF598•ATPase activity of the ASCC3 helicase is needed to split the leading stalled ribosome•ASCC generates 60S-nascent chains targeted by the ribosome quality control complex•Dispatch of the lead ribosome by ASCC allows trailing ribosomes to resume translation
Ribosomes translate mRNAs at ~4 to 6 codons per second. Various mRNA defects cause a ribosome to slow, leading to collisions with trailing ribosomes. Juszkiewicz et al. show that the ASC-1 complex containing the helicase ASCC3 selectively dissociates the lead ribosome of a collision, allowing trailing ribosomes to continue translation.
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.
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•RECQL5 is a general RNAPII elongation factor•RECQL5 reduces the elongation rate while decreasing pausing and arrest events•Loss of RECQL5 results in genome instability in genes and at common fragile sites•Incidents of genome instability colocalize with pause and arrest events
Rapid elongation by RNA polymerase II leads to increased transcriptional stress including a high incidence of pausing and arrests, which correlates with sites of genomic instability. RECQL5 modulates the rate of transcription, mitigating both the stress and instability effects.
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.
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•Def1 protein is proteolytically processed in response to transcription stress•Processing is ubiquitin and proteasome dependent•Processed Def1 (pr-Def1) accumulates in the nucleus, where it binds RNAPII•Using its CUE domain, pr-Def1 then recruits Elongin-Cullin via Ela1’s UbH domain
DNA damage induces proteasome-dependent proteolytic processing of Def1, releasing an activated protein fragment, which highlights how irreversible proteasome-mediated cleavage of a protein can act as a posttranslational modification in this signaling pathway.
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.
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•RQC factors involved in disassembling collided ribosomes suppress the cGAS pathway•Ribosomes interact with cGAS, stimulating its DNA-dependent activity•cGAS preferentially interacts with collided ribosomes•Ribosome collision leads to re-localization of cGAS to the cytosol and ISG activation
Wan et al. show that cGAS, a well-known DNA sensor, can also sense translation stress by direct interaction with ribosomes, which in turn induces accumulation of cGAS in the cytosol, stimulation of its DNA-dependent catalytic activity, and activation of innate immunity signaling via ISG activation.
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