Expression of human RECQL5 in Saccharomyces cerevisiae causes transcription defects and transcription-associated genome instability.

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.

DNA-directed termination of RNA polymerase II transcription.

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.

Transcription-Coupled Nucleotide Excision Repair and the Transcriptional Response to UV-Induced DNA Damage.

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.

Yeast Smy2 and its human homologs GIGYF1 and -2 regulate Cdc48/VCP function during transcription stress.

The "last resort" pathway results in ubiquitylation and degradation of RNA polymerase II in response to transcription stress and is governed by factors such as Def1 in yeast. Here, we show that the SMY2 gene acts as a multi-copy suppressor of DEF1 deletion and functions at multiple steps of the last resort pathway. We also provide genetic and biochemical evidence from disparate cellular processes that Smy2 works more broadly as a hitherto overlooked regulator of Cdc48 function. Similarly, the Smy2 homologs GIGYF1 and -2 affect the transcription stress response in human cells and regulate the function of the Cdc48 homolog VCP/p97, presently being explored as a target for cancer therapy. Indeed, we show that the apoptosis-inducing effect of VCP inhibitors NMS-873 and CB-5083 is GIGYF1/2 dependent.

An Important Role for RPRD1B in the Heat Shock Response.

During the heat shock response (HSR), heat shock factor (HSF1 in mammals) binds to target gene promoters, resulting in increased expression of heat shock proteins that help maintain protein homeostasis and ensure cell survival. Besides HSF1, only a relatively few transcription factors with a specific role in ensuring correctly regulated gene expression during the HSR have been described. Here, we use proteomic and genomic (CRISPR) screening to identify a role for RPRD1B in the response to heat shock. Indeed, cells depleted for RPRD1B are heat shock sensitive and show decreased expression of key heat shock proteins (HSPs). These results add to our understanding of the connection between basic gene expression mechanisms and the HSR.

UBAP2/UBAP2L regulate UV-induced ubiquitylation of RNA polymerase II and are the human orthologues of yeast Def1.

During transcription, RNA polymerase II (RNAPII) faces numerous obstacles, including DNA damage, which can lead to stalling or arrest. One mechanism to contend with this situation is ubiquitylation and degradation of the largest RNAPII subunit, RPB1 - the 'last resort' pathway. This conserved, multi-step pathway was first identified in yeast, and the functional human orthologues of all but one protein, RNAPII Degradation Factor 1 (Def1), have been discovered. Here we show that following UV-irradiation, human Ubiquitin-associated protein 2 (UBAP2) or its paralogue UBAP2-like (UBAP2L) are involved in the ubiquitylation and degradation of RNAPII through the recruitment of Elongin-Cul5 ubiquitin ligase. Together, our data indicate that UBAP2 and UBAP2L are the human orthologues of yeast Def1, and so identify the key missing proteins in the human last resort pathway.

Heat shock induces premature transcript termination and reconfigures the human transcriptome.

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.

Developmental regulation of neuronal gene expression by Elongator complex protein 1 dosage.

Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by a mutation in the Elongator complex protein 1 (ELP1) gene that leads to a tissue-specific reduction of ELP1 protein. Our work to generate a phenotypic mouse model for FD headed to the discovery that homozygous deletion of the mouse Elp1 gene leads to embryonic lethality prior to mid-gestation. Given that FD is caused by a reduction, not loss, of ELP1, we generated two new mouse models by introducing different copy numbers of the human FD ELP1 transgene into the Elp1 knockout mouse (Elp1-/-) and observed that human ELP1 expression rescues embryonic development in a dose-dependent manner. We then conducted a comprehensive transcriptome analysis in mouse embryos to identify genes and pathways whose expression correlates with the amount of ELP1. We found that ELP1 is essential for the expression of genes responsible for nervous system development. Further, gene length analysis of the differentially expressed genes showed that the loss of Elp1 mainly impacts the expression of long genes and that by gradually restoring Elongator, their expression is progressively rescued. Finally, through evaluation of co-expression modules, we identified gene sets with unique expression patterns that depended on ELP1 expression.

Elongation factor-specific capture of RNA polymerase II complexes.

Transcription of protein-coding genes is regulated by dynamic association of co-factors with RNA polymerase II (RNAPII). The function of these factors and their relationship with RNAPII is often poorly understood. Here, we present an approach for elongation-factor-specific mNET capture (ELCAP) of RNAPII complexes for sequencing and mass spectrometry analysis aimed at investigating the function of such RNAPII regulatory proteins. As proof of principle, we apply ELCAP to the RNAPII-associated proteins SCAF4 and SCAF8, which share an essential role as mRNA anti-terminators but have individual roles at the 3' end of genes. Mass spectrometry analysis shows that both SCAF4 and SCAF8 are part of RNAPII elongation complexes containing 3' end processing factors but depleted of splicing components. Importantly, the ELCAP sequencing (ELCAP-seq) profiles of SCAF4- and SCAF8-RNAPII complexes nicely reflect their function as mRNA-anti-terminators and their competing functions at the end of genes, where they prevent or promote transcriptional readthrough.

Transcription-coupled repair and the transcriptional response to UV-Irradiation.

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.

Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability.

Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA polymerase II, causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions. However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR-Cas9 screen, we identified the elongation factor ELOF1 as an important factor in the transcription stress response following DNA damage. We show that ELOF1 has an evolutionarily conserved role in transcription-coupled nucleotide excision repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair transcription-blocking lesions and resume transcription. Additionally, ELOF1 modulates transcription to protect cells against transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms.

Translation stress and collided ribosomes are co-activators of cGAS.

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.

Causes and consequences of RNA polymerase II stalling during transcript elongation.

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.

Annotation matters: validating the discovery of cancer drivers.

Advanced sequencing techniques have helped unveil numerous new, potential cancer driver mutations. However, manual curation and analysis of gene and protein annotation are essential to verify such discoveries. Our recent study of STK19 (Serine Threonine Kinase 19), a previously identified melanoma driver, is a clear example of the importance of such detailed analysis, with both STK19 gene and protein annotations in frequently used databases having been proven incorrect.

CDK13 cooperates with CDK12 to control global RNA polymerase II processivity.

The RNA polymerase II (POLII)-driven transcription cycle is tightly regulated at distinct checkpoints by cyclin-dependent kinases (CDKs) and their cognate cyclins. The molecular events underpinning transcriptional elongation, processivity, and the CDK-cyclin pair(s) involved remain poorly understood. Using CRISPR-Cas9 homology-directed repair, we generated analog-sensitive kinase variants of CDK12 and CDK13 to probe their individual and shared biological and molecular roles. Single inhibition of CDK12 or CDK13 induced transcriptional responses associated with cellular growth signaling pathways and/or DNA damage, with minimal effects on cell viability. In contrast, dual kinase inhibition potently induced cell death, which was associated with extensive genome-wide transcriptional changes including widespread use of alternative 3' polyadenylation sites. At the molecular level, dual kinase inhibition resulted in the loss of POLII CTD phosphorylation and greatly reduced POLII elongation rates and processivity. These data define substantial redundancy between CDK12 and CDK13 and identify both as fundamental regulators of global POLII processivity and transcription elongation.

Multiplex Cell Fate Tracking by Flow Cytometry.

Measuring differences in cell cycle progression is often essential to understand cell behavior under different conditions, treatments and environmental changes. Cell synchronization is widely used for this purpose, but unfortunately, there are many cases where synchronization is not an option. Many cell lines, patient samples or primary cells cannot be synchronized, and most synchronization methods involve exposing the cells to stress, which makes the method incompatible with the study of stress responses such as DNA damage. The use of dual-pulse labelling using EdU and BrdU can potentially overcome these problems, but the need for individual sample processing may introduce a great variability in the results and their interpretation. Here, we describe a method to analyze cell proliferation and cell cycle progression by double staining with thymidine analogues in combination with fluorescent cell barcoding, which allows one to multiplex the study and reduces the variability due to individual sample staining, reducing also the cost of the experiment.

DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1.

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.

The ASC-1 Complex Disassembles Collided Ribosomes.

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.

Evidence That STK19 Is Not an NRAS-dependent Melanoma Driver.

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.