Disruption of nuclear speckle integrity dysregulates RNA splicing in C9ORF72-FTD/ALS.

Expansion of an intronic (GGGGCC)n repeat within the C9ORF72 gene is the most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (C9-FTD/ALS), characterized with aberrant repeat RNA foci and noncanonical translation-produced dipeptide repeat (DPR) protein inclusions. Here, we elucidate that the (GGGGCC)n repeat RNA co-localizes with nuclear speckles and alters their phase separation properties and granule dynamics. Moreover, the essential nuclear speckle scaffold protein SRRM2 is sequestered into the poly-GR cytoplasmic inclusions in the C9-FTD/ALS mouse model and patient postmortem tissues, exacerbating the nuclear speckle dysfunction. Impaired nuclear speckle integrity induces global exon skipping and intron retention in human iPSC-derived neurons and causes neuronal toxicity. Similar alternative splicing changes can be found in C9-FTD/ALS patient postmortem tissues. This work identified novel molecular mechanisms of global RNA splicing defects caused by impaired nuclear speckle function in C9-FTD/ALS and revealed novel potential biomarkers or therapeutic targets.

PRPF40A induces inclusion of exons in GC-rich regions important for human myeloid cell differentiation.

We characterized the regulatory mechanisms and role in human myeloid cell survival and differentiation of PRPF40A, a splicing factor lacking a canonical RNA Binding Domain. Upon PRPF40A knockdown, HL-60 cells displayed increased cell death, decreased proliferation and slight differentiation phenotype with upregulation of immune activation genes. Suggestive of both redundant and specific functions, cell death but not proliferation was rescued by overexpression of its paralog PRPF40B. Transcriptomic analysis revealed the predominant role of PRPF40A as an activator of cassette exon inclusion of functionally relevant splicing events. Mechanistically, the exons exclusively upregulated by PRPF40A are flanked by short and GC-rich introns which tend to localize to nuclear speckles in the nucleus center. These PRPF40A regulatory features are shared with other splicing regulators such as SRRM2, SON, PCBP1/2, and to a lesser extent TRA2B and SRSF2, as a part of a functional network that regulates splicing partly via co-localization in the nucleus.

Short-term health effects of an urban regeneration programme in deprived neighbourhoods of Barcelona.

Urban regeneration programmes are interventions meant to enhance the wellbeing of residents in deprived areas, although empirical evidence reports mixed results. We evaluated the health impact of a participatory and neighbourhood-wide urban regeneration programme, Pla de Barris 2016-2020, in Barcelona. A pre-post with a comparison group study design. Using data from a cross-sectional survey performed in 2016 and 2021. The health outcomes analysed were mental health, alcohol and psychotropic drug use, perceived health status, physical activity and obesity. Depending on the investment, two intervention groups were defined: moderate- and high-intensity intervention groups. The analysis combined difference-in-difference estimation with an inverse weighting derived from a propensity score to reduce potential biases. The impact of the intervention in percentages and its confidence interval were estimated with a linear probability model with clustered adjusted errors. The intervention had a positive impact on health outcomes in women in the high-intensity intervention group: a reduction of 15.5% in the relative frequency of those experiencing poor mental health, and of 21.7% in the relative frequency of those with poor self-perceived health; and an increase of 13.7% in the relative frequency of those doing physical activity. No positive impact was observed for men, but an increase of 10.3% in the relative frequency of those using psychotropic drugs in the high-intensity intervention group. This study shows positive short-term effects of the urban regeneration programme Pla de Barris 2016-2020 on health outcomes in women in the high-intensity intervention group. These results can guide future interventions in other areas.

A novel intronic variant in ROBO3 associated with horizontal gaze palsy with progressive scoliosis: case report and literature review.

Horizontal gaze palsy with progressive scoliosis (HGPPS) is a rare, autosomal recessive inherited disorder caused by mutations in ROBO3 gene. The clinical features of HGPPS include horizontal gaze palsy, progressive scoliosis, other oculomotor abnormalities such as strabismus and nystagmus. Whole-exome sequencing (WES) is used to diagnose rare Mendelian disorders, when routine standard tests have failed to make a formal pathological diagnosis. However, WES may identify variants of uncertain significance (VUS) that may add further ambiguity to the diagnosis. We report the case of a 4-year-old boy with horizontal gaze palsy, progressive scoliosis, microcephaly, and mild developmental delay. WES identified an intronic VUS in ROBO3 gene. We performed minigene splicing functional analysis to confirm the pathogenicity of this VUS. This report illustrates that WES data analysis with supportive functional analysis provides an effective approach to improve the diagnostic yield for unsolved clinical cases. This case also highlights the phenotypic heterogeneity in patients with HGPPS.

Reconsidering the Use of Population Health Surveys for Monitoring of Mental Health.

Monitoring of the mental health status of the population and assessment of its determinants are 2 of the most relevant pillars of public mental health, and data from population health surveys could be instrumental to support them. Although these surveys could be an important and suitable resource for these purposes, due to different limitations and challenges, they are often relegated to the background behind other data sources, such as electronic health records. These limitations and challenges include those related to measurement properties and cross-cultural validity of the tools used for the assessment of mental disorders, their degree of representativeness, and possible difficulties in the linkage with other data sources. Successfully addressing these limitations could significantly increase the potential of health surveys in the monitoring of mental disorders and ultimately maximize the impact of the relevant policies to reduce their burden at the population level. The widespread use of data from population health surveys, ideally linked to electronic health records data, would enhance the quality of the information available for research, public mental health decision-making, and ultimately addressing the growing burden of mental disorders.

Incidence and Determinants of COVID-19 in Patients Seeking Treatment for Substance Use Disorder: A Patient-Based Linkage Study.

INTRODUCTION: People with substance use disorder (SUD) may be at increased risk of COVID-19 infection. However, there is little evidence regarding the incidence of and determinants associated with infection in this group. The aims of the study were to determine the cumulative incidence of COVID-19 among people who sought treatment for heroin, cocaine, cannabis, and alcohol use disorder in Catalonia; to identify sociodemographic, substance, and clinical determinants associated with COVID-19 infection among SUD patients; and to compare the cumulative incidence of COVID-19 infection in the population with SUD with that of the general population. METHODS: A patient-based retrospective observational study was conducted. The study population comprised people who sought treatment for heroin, cocaine, cannabis, or alcohol use disorder in Catalonia in 2018 and 2019. We analysed cumulative incidence of COVID-19 (confirmed by PCR test) from 25 February to 31 December 2020. Additionally, we used a log-link binomial generalized linear model for COVID-19 infection, using the substance as the exposition, adjusting for sociodemographic and clinical variables. RESULTS: Of the 23,092 individuals who sought treatment for SUD, 38.15% were considered suspected cases of COVID-19, and 2.60% (95% CI = 2.41-2.82) were confirmed positive for COVID-19 by PCR test during the study period. Those who sought treatment for alcohol use (cumulative incidence of COVID-19 of 3% [95% CI = 2.70-3.34]) had a higher risk ratio than, those who sought treatment for heroin use (cumulative incidence of 1.94% [95% CI = 1.47-2.56]). Being born outside of Spain, living in an institutionalized residence, having HIV, and being in a high morbidity group were associated with higher risk of COVID-19 infection. Meanwhile, the cumulative incidence of COVID-19 in the general population, according to public COVID-19 test data, was 3.86% (95% CI = 3.85-3.87). CONCLUSION: This study did not find higher cumulative incidence of COVID-19 infection among people with SUD in Catalonia in 2020, despite the clinical vulnerability of this population and their social disadvantage. However, differences were seen in the cumulative incidence of COVID-19 according to the substance for which treatment was sought. For example, those with alcohol dependence had a higher rate than those dependent on heroin. Further studies are needed to determine the factors contributing to these differences.

Implementation of the HepClink test-and-treat community strategy targeting Pakistani migrants with hepatitis C living in Catalonia (Spain) compared with the current practice of the Catalan health system: budget impact analysis.

OBJECTIVES: To perform a budget impact analysis of the HepClink test-and-treat strategy in which community health agents offer hepatitis C virus (HCV) testing, diagnosis and treatment to the Pakistani population living in Catalonia compared with the current practice of the Catalan health system (without targeted screening programmes). METHODS: We estimated the population of adult Pakistani migrants registered at the primary care centres in Catalonia by means of the Information System for the Development of Research in Primary Care (n=37 972 in 2019, Barcelona health area). This cohort was followed for a time period of 10 years after HCV diagnosis (2019-2028). The statistical significance of the differences observed in the anti-HCV positivity rate between screened and non-screened was confirmed (α=0.05). The budget impact was calculated from the perspective of the Catalan Department of Health. Sensitivity analyses included different levels of participation in HepClink: pessimistic, optimistic and maximum. RESULTS: The HepClink scenario screened a higher percentage of individuals (69.8%) compared with the current scenario of HCV care (39.7%). Viraemia was lower in the HepClink scenario compared with the current scenario (1.7% vs 2.5%, respectively). The budget impact of the HepClink scenario was €884 244.42 in 10 years. CONCLUSIONS: Scaling up the HepClink strategy to the whole Catalan territory infers a high budget impact for the Department of Health and allows increasing the detection of viraemia (+17.8%) among Pakistani migrants ≥18 years. To achieve a sustainable elimination of HCV by improving screening and treatment rates, there is room for improvement at two levels. First, taking advantage of the fact that 68.08% of the Pakistani population had visited their primary care physicians to reinforce targeted screening in primary care. Second, to use HepClink at the community level to reach individuals with reluctance to use healthcare services.

Reliability and cross-country equivalence of the 8-item version of the Patient Health Questionnaire (PHQ-8) for the assessment of depression: results from 27 countries in Europe.

Background: The 8-item version of the Patient Health Questionnaire (PHQ-8) is one of the self-reported questionnaires most frequently used worldwide for the screening and severity assessment of depression. However, in some European countries its reliability is unknown, and it is unclear whether its psychometric properties vary between European countries. Therefore, the aim of this study was to assess the internal structure, reliability and cross-country equivalence of the PHQ-8 in Europe. Methods: All participants from the 27 countries included in the second wave of the European Health Interview Survey (EHIS-2) between 2014 and 2015 with complete information on the PHQ-8 were included (n = 258,888). The internal structure of the PHQ-8 was assessed using confirmatory factor analyses (CFA) for categorical items. Additionally, the reliability of the questionnaire was assessed based on the internal consistency, Item Response Theory information functions, and item-discrimination (using Graded Response Models), and the cross-country equivalence based on multi-group CFA. Findings: The PHQ-8 shows high internal consistency for all countries. The countries in which the PHQ-8 was more reliable were Romania, Bulgaria and Cyprus and less reliable were Iceland, Norway and Austria. The PHQ-8 item with highest discrimination was item 2 (feeling down, depressed, or hopeless) in 24 of the 27 countries. Measurement invariance between countries in Europe was observed from multigroup CFA at the configural, metric and scalar levels. Interpretation: The results from our study, likely the largest study to the date assessing the internal structure, reliability and cross-country comparability of a self-reported mental health assessment measure, shows that the PHQ-8 has an adequate reliability and cross-country equivalence across the 27 European countries included. These results highlight the suitability of the comparisons of the PHQ-8 scores in Europe. They could be helpful to improve the screening and severity assessment of depressive symptoms at the European level. Funding: This work was partially funded by CIBER Epidemiology and Public Health (CIBERESP) as part of the Intramural call of 2021 (ESP21PI05).

Tobacco cessation among smokers under substance use treatment for alcohol and/or cannabis: study protocol and pilot study.

BACKGROUND: Approximately 80% of people with a substance use disorder (SUD) are smokers. Starting SUD treatment offers the opportunity to also quit smoking. The ACT-ATAC project aims to identify the predictors associated with smoking cessation among persons treated for alcohol and/or cannabis use disorder in Barcelona. This manuscript reports its methodology and the experience of carrying it out during the COVID-19 pandemic. METHODS: Mixed methods project with three substudies. Substudy 1 (S1) comprises heterogeneous discussion groups among clinicians. S2 has two prospective cohorts composed of smokers under treatment for alcohol and/or cannabis use disorder and the clinicians in charge of these patients. Participating smokers will be followed for 12 months and interviewed about their substance use and the tobacco cessation services received using the Spanish version of the users' Knowledge, Attitudes, and Services (S-KAS) scale. The clinicians will be asked about their self-reported practices in smoking cessation using the Knowledge, Attitudes, and Practices (S-KAP) scale. S3 comprises heterogeneous discussion groups with smokers. Data will be triangulated using qualitative and quantitative analyses. To facilitate the recruitment process, the researchers have introduced several strategies (design clear protocols, set monthly online meetings, extend the project, provide gift cards, etc.). DISCUSSION: The results of S1 were used to develop the questionnaires. S2 required some adjustments due to the COVID-19 pandemic, particularly the follow-up interviews being conducted by phone instead of face-to-face, and the recruitment rhythm was lower than expected. Recruitment will last until reaching at least 200-250 users. The fieldwork could not have been possible without the collaboration of the ACT-ATAC team and the introduction of several strategies. Trial registration The ACT-ATAC project has been successfully registered at Clinicaltrials.gov [NCT04841655].

Trends and inequalities in (healthy) life expectancy by neighbourhood during the COVID-19 epidemic in Barcelona.

OBJECTIVE: To analyse the trend in life expectancy (LE), healthy life expectancy (HLE) and socio-economic inequalities by neighbourhood in Barcelona from the pre-pandemic period (2018-2019) to the pandemic period (2020-2021). METHOD: LE and HLE at birth were computed using the municipal register of inhabitants and quality of life (EuroQol) from the Barcelona Health Survey of 2016. Inequalities were assessed with the gap between quantiles of neighbourhood income. RESULTS: In 2020, there was a reduction in LE among men (-1.98 years) and women (-2.44) and in HLE among men (-1.44). Socio-economic inequalities in LE and HLE between neighbourhoods widened since 2019 to 2021 (LE: from 3.92 to 4.86 years for men, and from 1.30 to 3.60 for women; HLE: from 6.88 to 7.70 years for men, and from 7.85 to 9.31 for women). CONCLUSIONS: The pandemic has substantially reduced LE and HLE, with larger effects among low-income neighbourhoods, especially among women.

SRRM2 organizes splicing condensates to regulate alternative splicing.

SRRM2 is a nuclear-speckle marker containing multiple disordered domains, whose dysfunction is associated with several human diseases. Using mainly EGFP-SRRM2 knock-in HEK293T cells, we show that SRRM2 forms biomolecular condensates satisfying most hallmarks of liquid-liquid phase separation, including spherical shape, dynamic rearrangement, coalescence and concentration dependence supported by in vitro experiments. Live-cell imaging shows that SRRM2 organizes nuclear speckles along the cell cycle. As bona-fide splicing factor present in spliceosome structures, SRRM2 deficiency induces skipping of cassette exons with short introns and weak splice sites, tending to change large protein domains. In THP-1 myeloid-like cells, SRRM2 depletion compromises cell viability, upregulates differentiation markers, and sensitizes cells to anti-leukemia drugs. SRRM2 induces a FES splice isoform that attenuates innate inflammatory responses, and MUC1 isoforms that undergo shedding with oncogenic properties. We conclude that SRRM2 acts as a scaffold to organize nuclear speckles, regulating alternative splicing in innate immunity and cell homeostasis.

ZAP isoforms regulate unfolded protein response and epithelial- mesenchymal transition.

Human ZAP inhibits many viruses, including HIV and coronaviruses, by binding to viral RNAs to promote their degradation and/or translation suppression. However, the regulatory role of ZAP in host mRNAs is largely unknown. Two major alternatively spliced ZAP isoforms, the constitutively expressed ZAPL and the infection-inducible ZAPS, play overlapping yet different antiviral and other roles that need further characterization. We found that the splicing factors hnRNPA1/A2, PTBP1/2, and U1-snRNP inhibit ZAPS production and demonstrated the feasibility to modulate the ZAPL/S balance by splice-switching antisense oligonucleotides in human cells. Transcriptomic analysis of ZAP-isoform-specific knockout cells revealed uncharacterized host mRNAs targeted by ZAPL/S with broad cellular functions such as unfolded protein response (UPR), epithelial-mesenchymal transition (EMT), and innate immunity. We established that endogenous ZAPL and ZAPS localize to membrane compartments and cytosol, respectively, and that the differential localization correlates with their target-RNA specificity. We showed that the ZAP isoforms regulated different UPR branches under resting and stress conditions and affected cell viability during ER stress. We also provided evidence for a different function of the ZAP isoforms in EMT-related cell migration, with effects that are cell-type dependent. Overall, this study demonstrates that the competition between splicing and IPA is a potential target for the modulation of the ZAPL/S balance, and reports new cellular transcripts and processes regulated by the ZAP isoforms.

Neighbourhood income inequalities in mental health in Barcelona 2001-2016: a Bayesian smoothed estimate.

OBJECTIVE: Obtaining reliable health estimates at the small area level (such as neighbourhoods) using survey data usually poses the problem of small sample sizes. To overcome this limitation, we explored smoothing techniques in order to estimate poor mental health prevalence at the neighbourhood level and analyse its profile by income in Barcelona city (Spain). METHOD: A Bayesian smoothing model with a logit-normal transformation was applied to four repeated cross-sectional waves of the Barcelona health survey for 2001, 2006, 2011 and 2016. Mental health status was identified from the 12-item General Health Questionnaire. Income inequalities were analysed with neighbourhood income in quantiles for each year and trends in the pooled analysis. RESULTS: The prevalence of poor mental health ranged from 14.6% in 2001 to 18.9% in 2016. The yearly difference between neighbourhoods was 12.4% in 2001, 16.7% in 2006, 14.2% in 2011, and 20.0% in 2016. The odds ratio and 95% credible interval (95%CI) of experiencing poor mental health was 1.40 times higher (95%CI: 1.02-1.91) in less advantaged neighbourhoods than in more advantaged neighbourhoods in 2001, 1.61 times higher (95%CI: 1.01-2.59) in 2006 and 2.31 times higher (95%CI: 1.57-3.40) in 2016. CONCLUSIONS: This study shows that the Bayesian smoothed techniques allows detection of inequalities in health in neighbourhoods and monitoring of interventions against them. In Barcelona, mental health problems are more prevalent in low-income neighbourhoods and raised in 2016.

Pan-cancer pervasive upregulation of 3' UTR splicing drives tumourigenesis.

Most mammalian genes generate messenger RNAs with variable untranslated regions (UTRs) that are important post-transcriptional regulators. In cancer, shortening at 3' UTR ends via alternative polyadenylation can activate oncogenes. However, internal 3' UTR splicing remains poorly understood as splicing studies have traditionally focused on protein-coding alterations. Here we systematically map the pan-cancer landscape of 3' UTR splicing and present this in SpUR ( http://www.cbrc.kaust.edu.sa/spur/home/ ). 3' UTR splicing is widespread, upregulated in cancers, correlated with poor prognosis and more prevalent in oncogenes. We show that antisense oligonucleotide-mediated inhibition of 3' UTR splicing efficiently reduces oncogene expression and impedes tumour progression. Notably, CTNNB1 3' UTR splicing is the most consistently dysregulated event across cancers. We validate its upregulation in hepatocellular carcinoma and colon adenocarcinoma, and show that the spliced 3' UTR variant is the predominant contributor to its oncogenic functions. Overall, our study highlights the importance of 3' UTR splicing in cancer and may launch new avenues for RNA-based anti-cancer therapeutics.

Inequalities in life expectancy by educational level and its decomposition in Barcelona, 2004-2018.

OBJECTIVE: To analyse the gap in life expectancy by educational level in the city of Barcelona from 2004 to 2018 and to decompose this gap by age and causes of death. METHOD: We computed abridged life tables at the age of 25 years by sex from 2004 to 2018 using standard methods. Educational level was categorised in two groups (lower secondary or less vs. upper secondary or higher education). The life expectancy gap was further decomposed by age and by causes of death based in Arriaga's method in 5-year age blocks up to the age of ≥ 90 years and broad causes of death using ICD-10 codes. RESULTS: The life expectancy gap at 25 years by educational level oscillated without trend at around 3.08 years for men and 1.93 years for women. Decomposition by age showed a favourable significant shift in the contribution to this gap from young to older ages for men, with few changes for women. Decomposition by causes of death showed that the diseases concentrating the largest share of the contribution were neoplasms and respiratory and circulatory disease. There was a significant downward trend in external causes for men and in infectious diseases for both men and women but a significant upward trend for respiratory disease for both sexes. CONCLUSIONS: The stability of the life expectancy gap by educational level during the period analysed resulted from a combination of divergent trends by age and causes of death among high and low educational levels.

Socioeconomic inequalities in self-assessed health and mental health in Barcelona, 2001-2016.

OBJECTIVE: Previous research has found persistent socioeconomic inequalities in health outcomes at the national level, with different patterns after the economic crisis. However, inequalities in urban areas are also important. This study analyses socioeconomic inequalities in self-assessed health and mental health in the city of Barcelona. METHOD: Repeated cross-sectional design using quinquennial data from the Barcelona Health Surveys carried out in 2001, 2006, 2011 and 2016 for the population older than 22 years. Robust Poisson regressions models were used to compute socioeconomic gradients and relative (RII) and slope indexes of inequality (SII) by occupational social class, with stratification by sex. RII and SII were also obtained with further adjustment by employment situation. RESULTS: A consistent socioeconomic gradient was found for all years except for 2011. Relative and absolute inequalities followed a V-shape, showing a drop during the economic crisis but widening thereafter to recover pre-crisis figures for self-assessed health and widening for mental health, in both relative and absolute terms in 2016. Adjustment for employment situation reduces inequalities but a large part of these inequalities remains, with variability across years. CONCLUSIONS: The lasting effects of the 2008 economic crisis and the austerity programmes imposed since then may have contributed to the persistence of socioeconomic inequalities in self-assessed health and the widening of those for mental health.

The lupus autoantigen La/Ssb is an Xist-binding protein involved in Xist folding and cloud formation.

Using the programmable RNA-sequence binding domain of the Pumilio protein, we FLAG-tagged Xist (inactivated X chromosome specific transcript) in live mouse cells. Affinity pulldown coupled to mass spectrometry was employed to identify a list of 138 candidate Xist-binding proteins, from which, Ssb (also known as the lupus autoantigen La) was validated as a protein functionally critical for X chromosome inactivation (XCI). Extensive XCI defects were detected in Ssb knockdown cells, including chromatin compaction, death of female mouse embryonic stem cells during in vitro differentiation and chromosome-wide monoallelic gene expression pattern. Live-cell imaging of Xist RNA reveals the defining XCI defect: Xist cloud formation. Ssb is a ubiquitous and versatile RNA-binding protein with RNA chaperone and RNA helicase activities. Functional dissection of Ssb shows that the RNA chaperone domain plays critical roles in XCI. In Ssb knockdown cells, Xist transcripts are unstable and misfolded. These results show that Ssb is critically involved in XCI, possibly as a protein regulating the in-cell structure of Xist.

Chromatin loop anchors predict transcript and exon usage.

Epigenomics and transcriptomics data from high-throughput sequencing techniques such as RNA-seq and ChIP-seq have been successfully applied in predicting gene transcript expression. However, the locations of chromatin loops in the genome identified by techniques such as Chromatin Interaction Analysis with Paired End Tag sequencing (ChIA-PET) have never been used for prediction tasks. Here, we developed machine learning models to investigate if ChIA-PET could contribute to transcript and exon usage prediction. In doing so, we used a large set of transcription factors as well as ChIA-PET data. We developed different Gradient Boosting Trees models according to the different tasks with the integrated datasets from three cell lines, including GM12878, HeLaS3 and K562. We validated the models via 10-fold cross validation, chromosome-split validation and cross-cell validation. Our results show that both transcript and splicing-derived exon usage can be effectively predicted with at least 0.7512 and 0.7459 of accuracy, respectively, on all cell lines from all kinds of validations. Examining the predictive features, we found that RNA Polymerase II ChIA-PET was one of the most important features in both transcript and exon usage prediction, suggesting that chromatin loop anchors are predictive of both transcript and exon usage.

Precarious Employment and Stress: The Biomedical Embodiment of Social Factors. PRESSED Project Study Protocol.

The PRESSED project aims to explain the links between a multidimensional measure of precarious employment and stress and health. Studies on social epidemiology have found a clear positive association between precarious employment and health, but the pathways and mechanisms to explain such a relationship are not well-understood. This project aims to fill this gap from an interdisciplinary perspective, integrating the social and biomedical standpoints to comprehensively address the complex web of consequences of precarious employment and its effects on workers' stress, health and well-being, including health inequalities. The project objectives are: (1) to analyze the association between multidimensional precarious employment and chronic stress among salaried workers in Barcelona, measured both subjectively and using biological indicators; (2) to improve our understanding of the pathways and mechanisms linking precarious employment with stress, health and well-being; and (3) to analyze health inequalities by gender, social class and place of origin for the first two objectives. The study follows a sequential mixed design. First, secondary data from the 2017 Survey on Workers and the Unemployed of Barcelona is analyzed (N = 1,264), yielding a social map of precarious employment in Barcelona that allows the contextualization of the scope and characteristics of this phenomenon. Drawing on these results, a second survey on a smaller sample (N = 255) on precarious employment, social precariousness and stress is envisaged. This study population is also asked to provide a hair sample to have their levels of cortisol and its related components, biomarkers of chronic stress, analyzed. Third, a sub-sample of the latter survey (n = 25) is selected to perform qualitative semi-structured interviews. This allows going into greater depth into how and why the experience of uncertainty, the precarization of living conditions, and the degradation of working conditions go hand-in-hand with precarious employment and have an impact on stress, as well as to explore the potential role of social support networks in mitigating these effects.

METTL4 catalyzes m6Am methylation in U2 snRNA to regulate pre-mRNA splicing.

N 6-methylation of 2'-O-methyladenosine (Am) in RNA occurs in eukaryotic cells to generate N6,2'-O-dimethyladenosine (m6Am). Identification of the methyltransferase responsible for m6Am catalysis has accelerated studies on the function of m6Am in RNA processing. While m6Am is generally found in the first transcribed nucleotide of mRNAs, the modification is also found internally within U2 snRNA. However, the writer required for catalyzing internal m6Am formation had remained elusive. By sequencing transcriptome-wide RNA methylation at single-base-resolution, we identified human METTL4 as the writer that directly methylates Am at U2 snRNA position 30 into m6Am. We found that METTL4 localizes to the nucleus and its conserved methyltransferase catalytic site is required for U2 snRNA methylation. By sequencing human cells with overexpressed Mettl4, we determined METTL4's in vivo target RNA motif specificity. In the absence of Mettl4 in human cells, U2 snRNA lacks m6Am thereby affecting a subset of splicing events that exhibit specific features such as 3' splice-site weakness and an increase in exon inclusion. These findings suggest that METTL4 methylation of U2 snRNA regulates splicing of specific pre-mRNA transcripts.