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Precursor mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans and defects in global pre-mRNA splicing associated with age are reduced by dietary restriction via splicing factor 1. Splicing factor 1 overexpression extends lifespan The collapse of protein homeostasis and increasing error rates in transcription are key risk factors for various chronic diseases, and are associated with ageing. William Mair and colleagues demonstrate that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans , and that defects in global pre-mRNA splicing associated with age are reduced by dietary restriction via splicing factor 1 (SFA-1). SFA-1 is specifically required for lifespan extension both by dietary restriction, and modulation of TORC1 pathway components. Overexpression of SFA-1 is sufficient to extend lifespan. This work suggests that modulation of specific spliceosome components can promote healthy ageing. Ageing is driven by a loss of transcriptional and protein homeostasis 1 , 2 , 3 and is the key risk factor for multiple chronic diseases. Interventions that attenuate or reverse systemic dysfunction associated with age therefore have the potential to reduce overall disease risk in the elderly. Precursor mRNA (pre-mRNA) splicing is a fundamental link between gene expression and the proteome, and deregulation of the splicing machinery is linked to several age-related chronic illnesses 4 , 5 . However, the role of splicing homeostasis in healthy ageing remains unclear. Here we demonstrate that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans . Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or subjected to dietary restriction, we find defects in global pre-mRNA splicing with age that are reduced by dietary restriction via splicing factor 1 (SFA-1; the C. elegans homologue of SF1, also known as branchpoint binding protein, BBP). We show that SFA-1 is specifically required for lifespan extension by dietary restriction and by modulation of the TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 kinase. We also demonstrate that overexpression of SFA-1 is sufficient to extend lifespan. Together, these data demonstrate a role for RNA splicing homeostasis in dietary restriction longevity and suggest that modulation of specific spliceosome components may prolong healthy ageing.

Background: Disseminated cutaneous malignant melanoma (CMM) is commonly unresponsive to standard chemotherapies, and there are as yet no predictive markers of therapy response. Methods: In the present study we collected fresh-frozen pretreatment lymph-node metastasis samples ( n =14) from melanoma patients with differential response to dacarbazine (DTIC) or temozolomide (TMZ) chemotherapy, to identify proteins with an impact on treatment response. We performed quantitative protein profiling using tandem mass spectrometry and compared the proteome differences between responders (R) and non-responders (NR), matched for age, gender and histopathological type of CMM. Results: Biological pathway analyses showed several signalling pathways differing between R vs NR, including Rho signalling. Gene expression profiling data was available for a subset of the samples, and the results were compared with the proteomics data. Four proteins with differential expression between R and NR were selected for technical validation by immunoblotting (ISYNA1, F13A1, CSTB and S100A13), and CSTB and S100A13 were further validated on a larger sample set by immunohistochemistry ( n =48). The calcium binding protein S100A13 was found to be significantly overexpressed in NR compared with R in all analyses performed. Conclusions: Our results suggest that S100A13 is involved in CMM resistance to DTIC/TMZ.

Phenylketonuria (PKU), one of the most common inherited diseases of amino acid metabolism, is caused by mutations in the phenylalanine hydroxylase (PAH) gene. Recently, PAH exon 11 was identified as a vulnerable exon due to a weak 3' splice site, with different exonic mutations affecting exon 11 splicing through disruption of exonic splicing regulatory elements. In this study, we report a novel intron 11 regulatory element, which is involved in exon 11 splicing, as revealed by the investigated pathogenic effect of variants c.1199+17G>A and c.1199+20G>C, identified in PKU patients. Both mutations cause exon 11 skipping in a minigene system. RNA binding assays indicate that binding of U1snRNP70 to this intronic region is disrupted, concomitant with a slightly increased binding of inhibitors hnRNPA1/2. We have investigated the effect of deletions and point mutations, as well as overexpression of adapted U1snRNA to show that this splicing regulatory motif is important for regulation of correct splicing at the natural 5' splice site. The results indicate that U1snRNP binding downstream of the natural 5' splice site determines efficient exon 11 splicing, thus providing a basis for development of therapeutic strategies to correct PAH exon 11 splicing mutations. In this work, we expand the functional effects of non-canonical intronic U1 snRNP binding by showing that it may enhance exon definition and that, consequently, intronic mutations may cause exon skipping by a novel mechanism, where they disrupt stimulatory U1 snRNP binding close to the 5' splice site. Notably, our results provide further understanding of the reported therapeutic effect of exon specific U1 snRNA for splicing mutations in disease.

BACKGROUND: Objectively measured physical activity between older individuals and between populations has been poorly described. We aimed to describe and compare the variation in accelerometry data in older UK (EPIC-Norfolk) and American (NHANES) adults. METHODS: Physical activity was measured by uniaxial accelerometry in 4,052 UK (49–91 years) and 3459 US older adults (49–85 years). We summarized physical activity as volume (average counts/minute), its underlying intensity distribution, and as time spent <100counts/minute, ≥809counts/minute and ≥2020counts/minute both for total activity and that undertaken in ≥10-min bouts. RESULTS: In EPIC-Norfolk 65 % of wear-time was spent at <100 counts/minute and 20 % spent in the range 100–500 counts/minute. Only 4.1 % of this cohort accumulated more than 30 min/day of activity above 2020 counts/minute in 10-min bouts. If a cut-point of >809 counts/minute is used 18.7 % of people reached the 30 min/day threshold. By comparison, 2.5 % and 9.5 % of American older adults accumulated activity at these levels, respectively. CONCLUSION: As assessed by objectively measured physical activity, the majority of older adults in this UK study did not meet current activity guidelines. Older adults in the UK were more active overall, but also spent more time being sedentary than US adults.

Background/Objectives: Fatness and fitness are associated with physical activity (PA) but less is known about the prospective associations of adiposity and muscle strength with PA. This study aimed to determine longitudinal associations of body mass index (BMI), waist circumference (WC) and grip strength (GS) with objectively measured PA. Subjects/Methods: Data are from the UK Biobank study. At baseline (2006–2010), BMI, WC and GS were objectively measured. At follow-up (2013–2015), a sub-sample of 93 015 participants (52 161 women) wore a tri-axial accelerometer on the dominant wrist for 7 days. Linear regression was performed to investigate longitudinal associations of standardised BMI, WC and GS at baseline with moderate-to-vigorous PA (MVPA) and acceleration after a median 5.7-years follow-up (interquartile range: 4.9–6.5 years). Results: Linear regression revealed strong inverse associations for BMI and WC, and positive associations for GS with follow-up PA; in women, MVPA ranges from lowest to highest quintiles of GS were 42–48 min day −1 in severely obese (BMI⩾35 kg m − 2 ), 52–57 min day −1 in obese (30⩽BMI<35 kg m − 2 ), 61–65 min day −1 in overweight (25⩽BMI<30 kg m − 2 ) and 69–75 min day −1 in normal weight (18.5⩽BMI<25 kg m −2 ). Follow-up MVPA was also lower in the lowest GS quintile (42–69 min day −1 ) compared with the highest GS quintile (48–75 min day −1 ) across BMI categories in women. The pattern of these associations was generally consistent for men, and in analyses using WC and mean acceleration as exposure and outcome, respectively. Conclusions: More pronounced obesity and poor strength at baseline independently predict lower activity levels at follow-up. Interventions and policies should aim to improve body composition and muscle strength to promote active living.

Oculocutaneous albinism (OCA) is a genetically heterogeneous disorder. Six genes are associated with autosomal recessive OCA ( TYR , OCA2 , TYRP1 , SLC45A2 , SLC24A5 and LRMDA ), and one gene, GPR143 , is associated with X-linked ocular albinism (OA). Molecular genetic analysis provides a genetic diagnosis in approximately 60% of individuals with clinical OA/OCA. A considerably number of the remaining 40% are heterozygous for a causative sequence variation in TYR . To identify missing causative sequence variants in these, we used a NGS based approach, genotyping and segregation analysis. We report two putative pathogenic haplotypes which only differ by two extremely rare SNVs, indicating that the haplotypes have a common derivation. Both haplotypes segregate consistent with an autosomal recessive inheritance pattern and include the allele p.S192Y-p.R402Q. An explanation for the pathogenicity of the haplotypes could be the combination of p.S192Y and p.R402Q. Homozygosity for the pathogenic haplotypes causes a partial albinism phenotype. In our cohort, 15% of affected individuals had a molecular genetic diagnosis involving the pathogenic haplotype. Consequently, the prevalence of albinism seems to be substantially underestimated, and children with unexplained bilateral subnormal vision and/or nystagmus should be analysed clinically and molecularly for albinism.

Background Many pathogenic genetic variants have been shown to disrupt mRNA splicing. Besides splice mutations in the well-conserved splice sites, mutations in splicing regulatory elements (SREs) may deregulate splicing and cause disease. A promising therapeutic approach is to compensate for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known. Results Here, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an iCLIP-identified hnRNP A1 binding site immediately downstream of the 5’ splice site. Because pseudoexons are well suited as models for constitutive exons which have been inactivated by pathogenic mutations in SREs, we used a pseudoexon in MTRR as a model and showed that an iCLIP-identified hnRNP A1 binding site downstream of the 5′ splice site can be blocked by SSOs to activate the exon. Conclusions The hnRNP A1 binding map can be used to identify potential targets for SSO-based therapy. Moreover, together with the hnRNP A1 consensus binding motif, the binding map may be used to predict whether disease-associated mutations and SNPs affect hnRNP A1 binding and eventually mRNA splicing.

The ataxia telangiectasia mutated and Rad3-related (ATR)-CHK1 pathway is the major signalling cascade activated in response to DNA replication stress. This pathway is associated with the core of the DNA replication machinery comprising CDC45, the replicative MCM2-7 hexamer, GINS (altogether forming the CMG complex), primase–polymerase (POLε, -α, and -δ) complex, and additional fork protection factors such as AND-1, CLASPIN (CLSPN), and TIMELESS/TIPIN. In this study, we report that functional protein kinase CK2α is critical for preserving replisome integrity and for mounting S-phase checkpoint signalling. We find that CDC45, CLSPN and MCM7 are novel CK2α interacting partners and these interactions are particularly important for maintenance of stable MCM7–CDC45, ATRIP–ATR–MCM7, and ATR–CLSPN protein complexes. Consistently, cells depleted of CK2α and treated with hydroxyurea display compromised replisome integrity, reduced chromatin binding of checkpoint mediator CLSPN, attenuated ATR-mediated S-phase checkpoint and delayed recovery of stalled forks. In further support of this, differential gene expression analysis by RNA-sequencing revealed that down-regulation of CK2α accompanies global shutdown of genes that are implicated in the S-phase checkpoint. These findings add to our understanding of the molecular mechanisms involved in DNA replication by showing that the protein kinase CK2α is essential for maintaining the stability of the replisome machinery and for optimizing ATR-CHK1 signalling activation upon replication stress.

Accurate quantification of physical activity energy expenditure is a key part of the effort to understand disorders of energy metabolism. The Actiheart, a combined heart rate (HR) and movement sensor, is designed to assess physical activity in populations. Objective: To examine aspects of Actiheart reliability and validity in mechanical settings and during walking and running. Methods: In eight Actiheart units, technical reliability (coefficients of variation, CV) and validity for movement were assessed with sinusoid accelerations (0.1–20 m/s 2 ) and for HR by simulated R-wave impulses (25–250 bpm). Agreement between Actiheart and ECG was determined during rest and treadmill locomotion (3.2–12.1 km/h). Walking and running intensity (in J/min/kg) was assessed with indirect calorimetry in 11 men and nine women (26–50 y, 20–29 kg/m 2 ) and modelled from movement, HR, and movement+HR by multiple linear regression, adjusting for sex. Results: Median intrainstrument CV was 0.5 and 0.03% for movement and HR, respectively. Corresponding interinstrument CV values were 5.7 and 0.03% with some evidence of heteroscedasticity for movement. The linear relationship between movement and acceleration was strong ( R 2 =0.99, P <0.001). Simulated R-waves were detected within 1 bpm from 30 to 250 bpm. The 95% limits of agreement between Actiheart and ECG were −4.2 to 4.3 bpm. Correlations with intensity were generally high ( R 2 >0.84, P <0.001) but significantly highest when combining HR and movement (SEE<1 MET). Conclusions: The Actiheart is technically reliable and valid. Walking and running intensity may be estimated accurately but further studies are needed to assess validity in other activities and during free-living. Sponsorship: The study received financial support from the Wellcome Trust and SB was supported by a scholarship from Unilever, UK.

Aims/hypothesis High levels of cardiorespiratory fitness (CRF) and physical activity (PA) are associated with a favourable metabolic risk profile. However, there has been no thorough exploration of the independent contributions of cardiorespiratory fitness and subcomponents of activity (total PA, time spent sedentary, and time spent in light, moderate and vigorous intensity PA) to metabolic risk factors in children and the relative importance of these factors. Methods We performed a population-based, cross-sectional study in 9- to 10- and 15- to 16-year-old boys and girls from three regions of Europe (n = 1709). We examined the independent associations of subcomponents of PA and CRF with metabolic risk factors (waist circumference, BP, fasting glucose, insulin, triacylglycerol and HDL-cholesterol levels). Clustered metabolic risk was expressed as a continuously distributed score calculated as the average of the standardised values of the six subcomponents. Results CRF (standardised β = -0.09, 95% CI -0.12, -0.06), total PA (standardised β = -0.08, 95% CI -0.10, -0.05) and all other subcomponents of PA were significantly associated with clustered metabolic risk. After excluding waist circumference from the summary score and further adjustment for waist circumference as a confounding factor, the magnitude of the association between CRF and clustered metabolic risk was attenuated (standardised β = -0.05, 95% CI -0.08, -0.02), whereas the association with total PA was unchanged (standardised β = -0.08 95% CI -0.10, -0.05). Conclusions/interpretation PA and CRF are separately and independently associated with individual and clustered metabolic risk factors in children. The association between CRF and clustered risk is partly mediated or confounded by adiposity, whereas the association between activity and clustered risk is independent of adiposity. Our results suggest that fitness and activity affect metabolic risk through different pathways.