Deficiency of FANCD2-associated nuclease KIAA1018/FAN1 sensitizes cells to interstrand crosslinking agents.

Cytotoxicity of cisplatin and mitomycin C (MMC) is ascribed largely to their ability to generate interstrand crosslinks (ICLs) in DNA, which block the progression of replication forks. The processing of ICLs requires the Fanconi anemia (FA) pathway, excision repair, and translesion DNA synthesis (TLS). It also requires homologous recombination (HR), which repairs double-strand breaks (DSBs) generated by cleavage of the blocked replication forks. Here we describe KIAA1018, an evolutionarily conserved protein that has an N-terminal ubiquitin-binding zinc finger (UBZ) and a C-terminal nuclease domain. KIAA1018 is a 5'-->3' exonuclease and a structure-specific endonuclease that preferentially incises 5' flaps. Like cells from FA patients, human cells depleted of KIAA1018 are sensitized to ICL-inducing agents and display chromosomal instability. The link of KIAA1018 to the FA pathway is further strengthened by its recruitment to DNA damage through interaction of its UBZ domain with monoubiquitylated FANCD2. We therefore propose to name KIAA1018 FANCD2-associated nuclease, FAN1.

Author Correction: Inflammatory memory sensitizes skin epithelial stem cells to tissue damage.

In Fig. 2g of this Article, a panel was inadvertently duplicated. The 'D30 IMQ' image was a duplicate of the 'D6 Ctrl' image. Fig. 2g has been corrected online to show the correct 'D30 IMQ' image (showing skin inflammation induced by the NALP3 agonist imiquimod, IMQ). The Supplementary Information to this Amendment contains the old, incorrect Fig. 2 for transparency.

Inflammatory memory sensitizes skin epithelial stem cells to tissue damage.

The skin barrier is the body's first line of defence against environmental assaults, and is maintained by epithelial stem cells (EpSCs). Despite the vulnerability of EpSCs to inflammatory pressures, neither the primary response to inflammation nor its enduring consequences are well understood. Here we report a prolonged memory to acute inflammation that enables mouse EpSCs to hasten barrier restoration after subsequent tissue damage. This functional adaptation does not require skin-resident macrophages or T cells. Instead, EpSCs maintain chromosomal accessibility at key stress response genes that are activated by the primary stimulus. Upon a secondary challenge, genes governed by these domains are transcribed rapidly. Fuelling this memory is Aim2, which encodes an activator of the inflammasome. The absence of AIM2 or its downstream effectors, caspase-1 and interleukin-1ß, erases the ability of EpSCs to recollect inflammation. Although EpSCs benefit from inflammatory tuning by heightening their responsiveness to subsequent stressors, this enhanced sensitivity probably increases their susceptibility to autoimmune and hyperproliferative disorders, including cancer.

Translation from unconventional 5' start sites drives tumour initiation.

We are just beginning to understand how translational control affects tumour initiation and malignancy. Here we use an epidermis-specific, in vivo ribosome profiling strategy to investigate the translational landscape during the transition from normal homeostasis to malignancy. Using a mouse model of inducible SOX2, which is broadly expressed in oncogenic RAS-associated cancers, we show that despite widespread reductions in translation and protein synthesis, certain oncogenic mRNAs are spared. During tumour initiation, the translational apparatus is redirected towards unconventional upstream initiation sites, enhancing the translational efficiency of oncogenic mRNAs. An in vivo RNA interference screen of translational regulators revealed that depletion of conventional eIF2 complexes has adverse effects on normal but not oncogenic growth. Conversely, the alternative initiation factor eIF2A is essential for cancer progression, during which it mediates initiation at these upstream sites, differentially skewing translation and protein expression. Our findings unveil a role for the translation of 5' untranslated regions in cancer, and expose new targets for therapeutic intervention.

Some like it translated: small ORFs in the 5'UTR.

The 5' untranslated region (5'UTR) is critical in determining post-transcriptional control, which is partly mediated by short upstream open reading frames (uORFs) present in half of mammalian transcripts. uORFs are generally considered to provide functionally important repression of the main-ORF by engaging initiating ribosomes, but under specific environmental conditions such as cellular stress, uORFs can become essential to activate the translation of the main coding sequence. In addition, a growing number of uORF-encoded bioactive microproteins have been described, which have the potential to significantly increase cellular protein diversity. Here we review the diverse cellular contexts in which uORFs play a critical role and discuss the molecular mechanisms underlying their function and regulation. The progress over the last decades in dissecting uORF function suggests that the 5'UTR remains an exciting frontier towards understanding how the cellular proteome is shaped in health and disease.

Translation of dipeptide repeat proteins from the C9ORF72 expanded repeat is associated with cellular stress.

Expansion of a hexanucleotide repeat (HRE), GGGGCC, in the C9ORF72 gene is recognized as the most common cause of familial amyotrophic lateral sclerosis (FALS), frontotemporal dementia (FTD) and ALS-FTD, as well as 5-10% of sporadic ALS. Despite the location of the HRE in the non-coding region (with respect to the main C9ORF72 gene product), dipeptide repeat proteins (DPRs) that are thought to be toxic are translated from the HRE in all three reading frames from both the sense and antisense transcript. Here, we identified a CUG that has a good Kozak consensus sequence as the translation initiation codon. Mutation of this CTG significantly suppressed polyglycine-alanine (GA) translation. GA was translated when the G4C2 construct was placed as the second cistron in a bicistronic construct. CRISPR/Cas9-induced knockout of a non-canonical translation initiation factor, eIF2A, impaired GA translation. Transfection of G4C2 constructs induced an integrated stress response (ISR), while triggering the ISR led to a continuation of translation of GA with a decline in conventional cap-dependent translation. These in vitro observations were confirmed in chick embryo neural cells. The findings suggest that DPRs translated from an HRE in C9ORF72 aggregate and lead to an ISR that then leads to continuing DPR production and aggregation, thereby creating a continuing pathogenic cycle.

HIF-1 antagonizes p53-mediated apoptosis through a secreted neuronal tyrosinase.

Hypoxia-inducible factor (HIF) is a transcription factor that regulates fundamental cellular processes in response to changes in oxygen concentration. HIFalpha protein levels are increased in most solid tumours and correlate with patient prognosis. The link between HIF and apoptosis, a major determinant of cancer progression and treatment outcome, is poorly understood. Here we show that Caenorhabditis elegans HIF-1 protects against DNA-damage-induced germ cell apoptosis by antagonizing the function of CEP-1, the homologue of the tumour suppressor p53. The antiapoptotic property of HIF-1 is mediated by means of transcriptional upregulation of the tyrosinase family member TYR-2 in the ASJ sensory neurons. TYR-2 is secreted by ASJ sensory neurons to antagonize CEP-1-dependent germline apoptosis. Knock down of the TYR-2 homologue TRP2 (also called DCT) in human melanoma cells similarly increases apoptosis, indicating an evolutionarily conserved function. Our findings identify a novel link between hypoxia and programmed cell death, and provide a paradigm for HIF-1 dictating apoptotic cell fate at a distance.

Apoptotic cell death under hypoxia.

Eukaryotic life depends largely on molecular oxygen. During evolution, ingenious mechanisms have evolved that allow organisms to adapt when oxygen levels decrease. Many of these adaptional responses to low oxygen are orchestrated by the heterodimeric transcription factor hypoxia-inducible factor (HIF). Here, we review the link between HIF and apoptosis.

No country for old methods: New tools for studying microproteins.

Microproteins encoded by small open reading frames (sORFs) have emerged as a fascinating frontier in genomics. Traditionally overlooked due to their small size, recent technological advancements such as ribosome profiling, mass spectrometry-based strategies and advanced computational approaches have led to the annotation of more than 7000 sORFs in the human genome. Despite the vast progress, only a tiny portion of these microproteins have been characterized and an important challenge in the field lies in identifying functionally relevant microproteins and understanding their role in different cellular contexts. In this review, we explore the recent advancements in sORF research, focusing on the new methodologies and computational approaches that have facilitated their identification and functional characterization. Leveraging these new tools hold great promise for dissecting the diverse cellular roles of microproteins and will ultimately pave the way for understanding their role in the pathogenesis of diseases and identifying new therapeutic targets.

In vivo CRISPR screens reveal SCAF1 and USP15 as drivers of pancreatic cancer.

Functionally characterizing the genetic alterations that drive pancreatic cancer is a prerequisite for precision medicine. Here, we perform somatic CRISPR/Cas9 mutagenesis screens to assess the transforming potential of 125 recurrently mutated pancreatic cancer genes, which revealed USP15 and SCAF1 as pancreatic tumor suppressors. Mechanistically, we find that USP15 functions in a haploinsufficient manner and that loss of USP15 or SCAF1 leads to reduced inflammatory TNFα, TGF-ß and IL6 responses and increased sensitivity to PARP inhibition and Gemcitabine. Furthermore, we find that loss of SCAF1 leads to the formation of a truncated, inactive USP15 isoform at the expense of full-length USP15, functionally coupling SCAF1 and USP15. Notably, USP15 and SCAF1 alterations are observed in 31% of pancreatic cancer patients. Our results highlight the utility of in vivo CRISPR screens to integrate human cancer genomics and mouse modeling for the discovery of cancer driver genes with potential prognostic and therapeutic implications.

Exploring the pan-cancer landscape of posttranscriptional regulation.

Understanding the mechanisms underlying cancer gene expression is critical for precision oncology. Posttranscriptional regulation is a key determinant of protein abundance and cancer cell behavior. However, to what extent posttranscriptional regulatory mechanisms impact protein levels and cancer progression is an ongoing question. Here, we exploit cancer proteogenomics data to systematically compare mRNA-protein correlations across 14 different human cancer types. We identify two clusters of genes with particularly low mRNA-protein correlations across all cancer types, shed light on the role of posttranscriptional regulation of cancer driver genes and drug targets, and unveil a cohort of 55 mutations that alter systems-wide posttranscriptional regulation. Surprisingly, we find that decreased levels of posttranscriptional control in patients correlate with shorter overall survival across multiple cancer types, prompting further mechanistic studies into how posttranscriptional regulation affects patient outcomes. Our findings underscore the importance of a comprehensive understanding of the posttranscriptional regulatory landscape for predicting cancer progression.

Monitoring the 5'UTR landscape reveals isoform switches to drive translational efficiencies in cancer.

Transcriptional and translational control are key determinants of gene expression, however, to what extent these two processes can be collectively coordinated is still poorly understood. Here, we use Nanopore long-read sequencing and cap analysis of gene expression (CAGE-seq) to document the landscape of 5' and 3' untranslated region (UTR) isoforms and transcription start sites of epidermal stem cells, wild-type keratinocytes and squamous cell carcinomas. Focusing on squamous cell carcinomas, we show that a small cohort of genes with alternative 5'UTR isoforms exhibit overall increased translational efficiencies and are enriched in ribosomal proteins and splicing factors. By combining polysome fractionations and CAGE-seq, we further characterize two of these UTR isoform genes with identical coding sequences and demonstrate that the underlying transcription start site heterogeneity frequently results in 5' terminal oligopyrimidine (TOP) and pyrimidine-rich translational element (PRTE) motif switches to drive mTORC1-dependent translation of the mRNA. Genome-wide, we show that highly translated squamous cell carcinoma transcripts switch towards increased use of 5'TOP and PRTE motifs, have generally shorter 5'UTRs and expose decreased RNA secondary structures. Notably, we found that the two 5'TOP motif-containing, but not the TOP-less, RPL21 transcript isoforms strongly correlated with overall survival in human head and neck squamous cell carcinoma patients. Our findings warrant isoform-specific analyses in human cancer datasets and suggest that switching between 5'UTR isoforms is an elegant and simple way to alter protein synthesis rates, set their sensitivity to the mTORC1-dependent nutrient-sensing pathway and direct the translational potential of an mRNA by the precise 5'UTR sequence.

Neutrophile-to-Lymphocyte Ratio (NLR) Identifies Patients with Coronavirus Infectious Disease 2019 (COVID-19) at High Risk for Deterioration and Mortality-A Retrospective, Monocentric Cohort Study.

Among people infected with SARS-CoV-2, the determination of clinical features associated with poor outcome is essential to identify those at high risk of deterioration. Here, we aimed to investigate clinical phenotypes of patients hospitalized due to COVID-19 and to examine the predictive value of the neutrophil-to-lymphocyte ratio (NLR) in a representative patient collective of the Swiss population. We conducted a retrospective monocentriccohort study with patients hospitalized due to COVID-19 between 27 February and 31 December 2020. Data were analyzed descriptively, using the binary logistic regression model, proportional odds logistic regression model, competing risk analysis, and summary measure analysis. A total of 454 patients were included in our study. Dyspnea, elevated respiratory rate, low oxygen saturation at baseline, age, and presence of multiple comorbidities were associated with a more severe course of the disease. A high NLR at baseline was significantly associated with disease severity, unfavorable outcome, and mortality. In non-survivors, NLR further increased during hospital stay, whereas in survivors, NLR decreased. In conclusion, our data emphasize the importance of accurate history taking and clinical examination upon admission and confirm the role of baseline NLR as a surrogate marker for increased disease severity, unfavorable outcome, and mortality in patients hospitalized due to infection with SARS-CoV-2.

External Validation of the Modified 4C Deterioration Model and 4C Mortality Score for COVID-19 Patients in a Swiss Tertiary Hospital.

Prognostic models to predict the deterioration and mortality risk in COVID-19 patients are utterly needed to assist in informed decision making. Most of these models, however, are at high risk of bias, model overfitting, and unclear reporting. Here, we aimed to externally validate the modified (urea was omitted) 4C Deterioration Model and 4C Mortality Score in a cohort of Swiss COVID-19 patients and, second, to evaluate whether the inclusion of the neutrophil-to-lymphocyte ratio (NLR) improves the predictive performance of the models. We conducted a retrospective single-centre study with adult patients hospitalized with COVID-19. Both prediction models were updated by including the NLR. Model performance was assessed via the models' discriminatory performance (area under the curve, AUC), calibration (intercept and slope), and their performance overall (Brier score). For the validation of the 4C Deterioration Model and Mortality Score, 546 and 527 patients were included, respectively. In total, 133 (24.4%) patients met the definition of in-hospital deterioration. Discrimination of the 4C Deterioration Model was AUC = 0.78 (95% CI 0.73-0.82). A total of 55 (10.44%) patients died in hospital. Discrimination of the 4C Mortality Score was AUC = 0.85 (95% CI 0.79-0.89). There was no evidence for an incremental value of the NLR. Our data confirm the role of the modified 4C Deterioration Model and Mortality Score as reliable prediction tools for the risk of deterioration and mortality. There was no evidence that the inclusion of NLR improved model performance.

The integrated stress response remodels the microtubule-organizing center to clear unfolded proteins following proteotoxic stress.

Cells encountering stressful situations activate the integrated stress response (ISR) pathway to limit protein synthesis and redirect translation to better cope. The ISR has also been implicated in cancers, but redundancies in the stress-sensing kinases that trigger the ISR have posed hurdles to dissecting physiological relevance. To overcome this challenge, we targeted the regulatory node of these kinases, namely, the S51 phosphorylation site of eukaryotic translation initiation factor eIF2α and genetically replaced eIF2α with eIF2α-S51A in mouse squamous cell carcinoma (SCC) stem cells of skin. While inconsequential under normal growth conditions, the vulnerability of this ISR-null state was unveiled when SCC stem cells experienced proteotoxic stress. Seeking mechanistic insights into the protective roles of the ISR, we combined ribosome profiling and functional approaches to identify and probe the functional importance of translational differences between ISR-competent and ISR-null SCC stem cells when exposed to proteotoxic stress. In doing so, we learned that the ISR redirects translation to centrosomal proteins that orchestrate the microtubule dynamics needed to efficiently concentrate unfolded proteins at the microtubule-organizing center so that they can be cleared by the perinuclear degradation machinery. Thus, rather than merely maintaining survival during proteotoxic stress, the ISR also functions in promoting cellular recovery once the stress has subsided. Remarkably, this molecular program is unique to transformed skin stem cells, hence exposing a vulnerability in cancer that could be exploited therapeutically.

An RNAi screen unravels the complexities of Rho GTPase networks in skin morphogenesis.

During mammalian embryogenesis, extensive cellular remodeling is needed for tissue morphogenesis. As effectors of cytoskeletal dynamics, Rho GTPases and their regulators are likely involved, but their daunting complexity has hindered progress in dissecting their functions. We overcome this hurdle by employing high throughput in utero RNAi-mediated screening to identify key Rho regulators of skin morphogenesis. Our screen unveiled hitherto unrecognized roles for Rho-mediated cytoskeletal remodeling events that impact hair follicle specification, differentiation, downgrowth and planar cell polarity. Coupling our top hit with gain/loss-of-function genetics, interactome proteomics and tissue imaging, we show that RHOU, an atypical Rho, governs the cytoskeletal-junction dynamics that establish columnar shape and planar cell polarity in epidermal progenitors. Conversely, RHOU downregulation is required to remodel to a conical cellular shape that enables hair bud invagination and downgrowth. Our findings underscore the power of coupling screens with proteomics to unravel the physiological significance of complex gene families.

MINA-1 and WAGO-4 are part of regulatory network coordinating germ cell death and RNAi in C. elegans.

Post-transcriptional control of mRNAs by RNA-binding proteins (RBPs) has a prominent role in the regulation of gene expression. RBPs interact with mRNAs to control their biogenesis, splicing, transport, localization, translation, and stability. Defects in such regulation can lead to a wide range of human diseases from neurological disorders to cancer. Many RBPs are conserved between Caenorhabditis elegans and humans, and several are known to regulate apoptosis in the adult C. elegans germ line. How these RBPs control apoptosis is, however, largely unknown. Here, we identify mina-1(C41G7.3) in a RNA interference-based screen as a novel regulator of apoptosis, which is exclusively expressed in the adult germ line. The absence of MINA-1 causes a dramatic increase in germ cell apoptosis, a reduction in brood size, and an impaired P granules organization and structure. In vivo crosslinking immunoprecipitation experiments revealed that MINA-1 binds a set of mRNAs coding for RBPs associated with germ cell development. Additionally, a system-wide analysis of a mina-1 deletion mutant compared with wild type, including quantitative proteome and transcriptome data, hints to a post-transcriptional regulatory RBP network driven by MINA-1 during germ cell development in C. elegans. In particular, we found that the germline-specific Argonaute WAGO-4 protein levels are increased in mina-1 mutant background. Phenotypic analysis of double mutant mina-1;wago-4 revealed that contemporary loss of MINA-1 and WAGO-4 strongly rescues the phenotypes observed in mina-1 mutant background. To strengthen this functional interaction, we found that upregulation of WAGO-4 in mina-1 mutant animals causes hypersensitivity to exogenous RNAi. Our comprehensive experimental approach allowed us to describe a phenocritical interaction between two RBPs controlling germ cell apoptosis and exogenous RNAi. These findings broaden our understanding of how RBPs can orchestrate different cellular events such as differentiation and death in C. elegans.

Direct in vivo RNAi screen unveils myosin IIa as a tumor suppressor of squamous cell carcinomas.

Mining modern genomics for cancer therapies is predicated on weeding out "bystander" alterations (nonconsequential mutations) and identifying "driver" mutations responsible for tumorigenesis and/or metastasis. We used a direct in vivo RNA interference (RNAi) strategy to screen for genes that upon repression predispose mice to squamous cell carcinomas (SCCs). Seven of our top hits-including Myh9, which encodes nonmuscle myosin IIa-have not been linked to tumor development, yet tissue-specific Myh9 RNAi and Myh9 knockout trigger invasive SCC formation on tumor-susceptible backgrounds. In human and mouse keratinocytes, myosin IIa's function is manifested not only in conventional actin-related processes but also in regulating posttranscriptional p53 stabilization. Myosin IIa is diminished in human SCCs with poor survival, which suggests that in vivo RNAi technology might be useful for identifying potent but low-penetrance tumor suppressors.

DEPDC1/LET-99 participates in an evolutionarily conserved pathway for anti-tubulin drug-induced apoptosis.

Microtubule-targeting chemotherapeutics induce apoptosis in cancer cells by promoting the phosphorylation and degradation of the anti-apoptotic BCL-2 family member MCL1. The signalling cascade linking microtubule disruption to MCL1 degradation remains however to be defined. Here, we establish an in vivo screening strategy in Caenorhabditis elegans to uncover genes involved in chemotherapy-induced apoptosis. Using an RNAi-based screen, we identify three genes required for vincristine-induced apoptosis. We show that the DEP domain protein LET-99 acts upstream of the heterotrimeric G protein alpha subunit GPA-11 to control activation of the stress kinase JNK-1. The human homologue of LET-99, DEPDC1, similarly regulates vincristine-induced cell death by promoting JNK-dependent degradation of the BCL-2 family protein MCL1. Collectively, these data uncover an evolutionarily conserved mediator of anti-tubulin drug-induced apoptosis and suggest that DEPDC1 levels could be an additional determinant for therapy response upstream of MCL1.

A novel mouse model for inhibition of DOHH-mediated hypusine modification reveals a crucial function in embryonic development, proliferation and oncogenic transformation.

The central importance of translational control by post-translational modification has spurred major interest in regulatory pathways that control translation. One such pathway uniquely adds hypusine to eukaryotic initiation factor 5A (eIF5A), and thereby affects protein synthesis and, subsequently, cellular proliferation through an unknown mechanism. Using a novel conditional knockout mouse model and a Caenorhabditis elegans knockout model, we found an evolutionarily conserved role for the DOHH-mediated second step of hypusine synthesis in early embryonic development. At the cellular level, we observed reduced proliferation and induction of senescence in 3T3 Dohh-/- cells as well as reduced capability for malignant transformation. Furthermore, mass spectrometry showed that deletion of DOHH results in an unexpected complete loss of hypusine modification. Our results provide new biological insight into the physiological roles of the second step of the hypusination of eIF5A. Moreover, the conditional mouse model presented here provides a powerful tool for manipulating hypusine modification in a temporal and spatial manner, to analyse both how this unique modification normally functions in vivo as well as how it contributes to different pathological conditions.