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bioRxiv2021

Chromosome specific telomere lengths and the minimal functional telomere revealed by nanopore sequencing

We developed a method to tag telomeres and measure telomere length by nanopore sequencing in the yeast S. cerevisiae. Nanopore allows long read sequencing through the telomere, subtelomere and into unique chromosomal sequence, enabling assignment of telomere length to a specific chromosome end. We observed chromosome end specific telomere lengths that were stable over 120 cell divisions. These stable chromosome specific telomere lengths may be explained by stochastic clonal variation or may represent a new biological mechanism that maintains equilibrium unique to each chromosomes end. We examined the role of RIF1 and TEL1 in telomere length regulation and found that TEL1 is epistatic to RIF1 at most telomeres, consistent with the literature. However, at telomeres that lack subtelomeric Y sequences, tel1{Delta} rif1{Delta} double mutants had a very small, but significant, increase in telomere length compared to the tel1{Delta} single mutant, suggesting an influence of Y elements on telomere length regulation. We sequenced telomeres in a telomerase-null mutant (est2{Delta}) and found the minimal telomere length to be around 75bp. In these est2{Delta} mutants there were many apparent telomere recombination events at individual telomeres before the generation of survivors, and these events were significantly reduced in est2{Delta} rad52{Delta} double mutants. The rate of telomere shortening in the absence of telomerase was similar across all chromosome ends at about 5 bp per generation. This new method gives quantitative, high resolution telomere length measurement at each individual chromosome end, suggests possible new biological mechanisms regulating telomere length, and provides capability to test new hypotheses.

molecular biology↗

bioRxiv2021

Deep assessment of human disease-associated ribosomal RNA modifications using Nanopore direct RNA sequencing

The catalytically active component of ribosomes, rRNA, is long studied and heavily modified. However, little is known about functional and pathological consequences of changes in human rRNA modification status. Direct RNA sequencing on the Nanopore platform enables the direct assessment of rRNA modifications. We established a targeted Nanopore direct rRNA sequencing approach and applied it to CRISPR-Cas9 engineered HCT116 cells, lacking specific enzymatic activities required to establish defined rRNA base modifications. We analyzed these sequencing data along with wild type samples and in vitro transcribed reference sequences to specifically detect changes in modification status. We show for the first time that direct RNA-sequencing is feasible on smaller, i.e. Flongle, flow cells. Our targeted approach reduces RNA input requirements, making it accessible to the analysis of limited samples such as patient derived material. The analysis of rRNA modifications during cardiomyocyte differentiation of human induced pluripotent stem cells, and of heart biopsies from cardiomyopathy patients revealed altered modifications of specific sites, among them pseudouridines, 2-O-methylation of riboses and acetylation of cytidines. Targeted direct rRNA-seq analysis with JACUSA2 opens up the possibility to analyze dynamic changes in rRNA modifications in a wide range of biological and clinical samples.

molecular biology↗

bioRxiv2022

Sequence assignment validation in cryo-EM models with checkMySequence

The availability of new AI-based protein structure prediction tools radically changed the way cryo-EM maps are interpreted, but it has not eliminated the challenges of map interpretation faced by a microscopist. Models will continue to be locally rebuilt and refined using interactive tools. This inevitably results in occasional errors, among which register-shifts remain one of the most difficult to identify and correct. Here we introduce checkMySequence; a fast, fully automated and parameter-free method for detecting register-shifts in protein models built into cryo-EM maps. We show that the method can assist model building in cases where poorer map resolution hinders visual interpretation. We also show that checkMySequence could have helped avoid a widely discussed sequence register error in a model of SARS-CoV-2 RNA-dependent RNA polymerase that was originally detected thanks to a visual residue-by-residue inspection by members of the structural biology community. SynopsisWe present a new method, checkMySequence, for fast and automated detection of register errors in protein models built into cryo-EM reconstructions.

molecular biology↗

bioRxiv2023

Determining how MAFA and MAFB transcription factors activity is influenced by structural differences predicted by AlphaFold2

MAFA and MAFB are related basic-leucine-zipper domain containing transcription factors which have important overlapping and distinct regulatory roles in a variety of cellular contexts, including hormone production in pancreatic islet and {beta} cells. Here we first examined how mutating conserved MAF protein-DNA contacts obtained from X-ray crystal structure analysis impacted their DNA-binding and Insulin enhancer-driven activity. While most of these interactions were essential and their disruption severely compromised activity, we identified that regions outside of the contact areas also contributed to activity. AlphaFold 2, an artificial intelligence-based structural prediction program, was next used to determine if there were also differences in the three-dimensional organization of the non-DNA binding/dimerization sequences of MAFA and MAFB. This analysis was conducted on the wildtype (WT) proteins as well as the pathogenic MAFASer64Phe and MAFBSer70Ala trans-activation domain mutants, with differences revealed between MAFAWT and MAFBWT as well as between MAFASer64Phe and MAFAWT, but not between MAFBSer70Ala and MAFBWT. Moreover, dissimilarities between these proteins were also observed in their ability to cooperatively stimulate Insulin enhancer-driven activity in the presence of other islet-enriched transcription factors. Analysis of MAFA and MAFB chimeras disclosed that these properties were greatly influenced by unique C-terminal region structural differences predicted by AlphaFold 2. Importantly, these results have revealed features of these closely related proteins that are functionally significant in islet biology.

molecular biology↗

bioRxiv2024

Age-Related Patterns of DNA Methylation Changes

Epigenetic clocks have achieved significant success in aging research, but they often assume linear methylation changes with age and lack biological interpretability. Using data from 4,641 samples across 23 GEO datasets, we analyzed 1,557 CpGs from nine widely used clocks with minimal overlap, and identified consistent age-associated methylation patterns. We then identified 19,432 age-associated CpGs (aaCpGs) that were strongly correlated with age and showed high consistency between sexes, with faster methylation changes observed in males. Most aaCpGs were identified during early and late life stages, indicating accelerated epigenetic changes during development and aging. No specific genomic enrichment was observed. Clustering analysis revealed four distinct, non-linear age-related methylation trajectories. These findings underscore the complexity of epigenetic aging and suggest that current clocks may overlook important dynamic patterns, particularly after age 65. Incorporating these insights could improve the accuracy and biological relevance of future epigenetic clocks, especially for use across diverse age ranges and populations.

molecular biology↗

bioRxiv2025

Site-1 Protease is a negative regulator of sarcolipin promoter activity

The timed contraction and relaxation of myofibers in tissues such as the heart and skeletal muscle occurs via the tightly regulated movement of calcium ions into and out of the sarcoplasmic reticulum (SR). In skeletal muscle, this phenomenon enables humans to exercise, perform day-to-day tasks, and to breathe. Sarcolipin, a small regulatory protein, prevents calcium ions from entering the SR by binding to and inhibiting SERCA, contributing to myofiber contraction. Disruptions in sarcolipin expression are implicated in the pathophysiology of obesity and musculoskeletal disease. However, the mechanisms regulating sarcolipin expression are not clearly understood. We recently showed that Site-1 Protease (S1P) is a regulator of skeletal muscle function and mass. Here, we report that deleting S1P in mouse skeletal muscle increases sarcolipin expression, without impacting calcium SR flux. In cultured cells, S1P negatively regulates sarcolipin by activating the transcription factor ATF6, which inhibits basal- and calcineurin-stimulated sarcolipin promoter activity. We identified a cAMP response element binding protein (CREB) binding site on the sarcolipin promoter that is necessary for promoter activation, and show that in muscle, CREB binds to the sarcolipin promoter and that this binding is enhanced when S1P is deleted. These discoveries expand our knowledge of S1P biology and the mechanisms controlling calcium regulatory genes.

molecular biology↗

bioRxiv2026

Endogenous Nuclear Desmin Associates with the Nuclear Envelope in Human Skeletal Muscle Cells

Desmin is the major intermediate filament protein of muscle, yet its potential nuclear functions remain poorly understood. Here, we investigated the nuclear localization and organization of endogenous desmin in human skeletal muscle cells using quantitative confocal microscopy, biochemical fractionation, targeted MRM/SRM proteomics, and computational structural modeling. Desmin localized to the nuclear compartment and exhibited significantly stronger colocalization with emerin and lamin A/C than with lamin B1. Nuclear fractionation and targeted proteomics independently confirmed endogenous nuclear desmin, while protein-protein docking predicted structurally plausible interactions with all three nuclear envelope proteins, supporting a context-dependent nuclear interaction network. Together, these findings identify the nuclear envelope as a functional platform for desmin and suggest that nuclear desmin contributes to nucleo-cytoskeletal communication and the organization of mechanically responsive nuclear architecture during myogenesis. This work expands the current understanding of desmin biology and provides a framework for investigating its role in desmin-related muscle disease.

molecular biology↗

bioRxiv2026

Biological Mechanisms of Strength Preservation During Calorie Restriction-Induced Weight Loss Among Young- to Middle-Aged Adults without Obesity

PurposeWeight loss is often pursued to improve cardiometabolic health and quality of life. However, rapid weight loss can lead to reductions in lean soft tissue mass and strength to compromise body composition and functional ability. Thus, identifying molecular predictors of muscular strength preservation during weight loss is critical to mitigating these effects. MethodsWe conducted a secondary analysis of the CALERIETM (Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy) trial, a two-year randomized controlled study of caloric restriction (CR) or ad-libitum intake in healthy adults without obesity. Among 198 participants, changes in whole-body mass and knee extensor strength were assessed over the first 12 months of the study which was primarily characterized by weight loss. Transcriptomic profiling was conducted in a subset of 42 participants who provided skeletal muscle samples. Linear regression was used to model the relationship between strength change and gene expression change, while controlling for changes in whole-body mass. Gene set enrichment analysis (GSEA) was performed using Hallmark pathways. Individual-level pathway analysis was performed via gene set variation analysis (GSVA). ResultsWe identified 96 out of 198 individuals (48.5%) who maintained or improved strength relative to body mass during weight loss (i.e. individuals with residuals > 0). Transcriptomics analysis on a subset of 42 individuals revealed 151 genes significantly associated with change in strength after accounting for change in whole-body mass (p < 0.01). Hub genes were identified as HSP90AA1 ({beta} = 34.45, SE = 7.32, p <0.001), EIF3A ({beta} = 36.14, SE = 10.27, p < 0.001), EIF5B ({beta} = 49.94, SE = 11.28, p < 0.001), and H3C1 ({beta} = -15.87, SE = 4.85, p < 0.001). GSEA revealed significant involvement of pathways related to cellular proliferation, immune regulation, protein secretion, and checkpoint control processes. GSVA identified a similar set of pathways. ConclusionsThese findings highlight molecular pathways supporting strength retention during CR-induced weight loss. Heat-shock protein, HSP90AA1, warrants further investigation as a candidate target for preserving muscle strength during interventions aimed at weight reduction.

molecular biology↗

bioRxiv2025

Assigning Targetable Molecular Pathways to Transdiagnostic Subgroups Across Autism and Related Neurodevelopmental Disorders

The heterogeneity of autism and related neurodevelopmental conditions has impeded accurate prognoses and treatment discovery. Using translational neuroimaging across 135 mouse models (3,515 mice) and two human MRI datasets (n = 1,234 and n = 1,015), we derived participant subgroups from shared neuroanatomical features. These subgroups did not distinguish autism, ADHD, or OCD diagnoses and only modestly differentiated cognitive and behavioural phenotypes. Instead, they mapped onto four molecular pathways: (1) synaptic function; (2) MAPK and Wnt signalling; (3) chromatin modification and cellular stress responses; and (4) broader chromatin, immune, and second-messenger signalling pathways. This framework bridges preclinical models and idiopathic human neurodevelopmental conditions, linking patients to biologically relevant molecular mechanisms.

neuroscience↗

bioRxiv2021

Development of a universal nanobody-binding Fab module for fiducial-assisted cryo- EM studies of membrane proteins

With conformation-specific nanobodies being used for a wide range of structural, biochemical, and cell biological applications, there is a demand for antigen-binding fragments (Fabs) that specifically and tightly bind these nanobodies without disturbing the nanobody-target protein interaction. Here we describe the development of a synthetic Fab (termed NabFab) that binds the scaffold of an alpaca-derived nanobody with picomolar affinity. We demonstrate that upon CDR grafting onto this parent nanobody scaffold, nanobodies recognizing diverse target proteins and derived from llama or camel can cross-react with NabFab without loss of affinity. Using NabFab as a fiducial and size enhancer (50 kDa), we determined the high-resolution cryo-EM structures of nanobody-bound VcNorM and ScaDMT, both small membrane proteins of ~50 kDa. Using an additional anti-Fab nanobody further facillitated reliable initial 3D structure determination from small cryo-EM test datasets. Given that NabFab is of synthetic origin, humanized, and can be conveniently expressed in E. coli in large amounts, it may not only be useful for structural biology, but also for biomedical applications.

molecular biology↗

bioRxiv2023

Comparative analyses of disease-linked missense mutations in the RNA exosome modeled in budding yeast reveal distinct functional consequences in translation

The RNA exosome is a multi-subunit, evolutionarily conserved ribonuclease complex that is essential for processing, decay and surveillance of many cellular RNAs. Missense mutations in genes encoding the structural subunits of the RNA exosome complex cause a diverse range of diseases, collectively known as RNA exosomopathies, often involving neurological and developmental defects. The varied symptoms suggest that different mutations lead to distinct in vivo consequences. To investigate these functional consequences and distinguish whether they are unique to each RNA exosomopathy mutation, we generated a collection of in vivo models by introducing pathogenic missense mutations in orthologous S. cerevisiae genes. Comparative RNA-seq analysis assessing broad transcriptomic changes in each mutant model revealed that three yeast mutant models, rrp4-G226D, rrp40-W195R and rrp46-L191H, which model mutations in the genes encoding EXOSC2, EXOSC3 and EXOSC5, respectively, had the largest transcriptomic differences. While some transcriptomic changes, particularly in transcripts related to ribosome biogenesis, were shared among mutant models, each mutation also induced unique transcriptomic changes. Thus, our data suggests that while there are some shared consequences, there are also distinct differences in RNA exosome function by each variant. Assessment of ribosome biogenesis and translation defects in the three models revealed distinct differences in polysome profiles. Collectively, our results provide the first comparative analyses of RNA exosomopathy mutant models and suggest that different RNA exosome gene mutations result in in vivo consequences that are both unique and shared across each variant, providing further insight into the biology underlying each distinct pathology.

molecular biology↗

bioRxiv2024

Chemical tools to define and manipulate interferon-inducible Ubl protease USP18

Ubiquitin-specific protease 18 (USP18) is a multifunctional cysteine protease primarily responsible for deconjugating interferon-inducible ubiquitin-like (Ubl) modifier ISG15 from protein substrates. Here, we report the design and synthesis of activity-based probes (ABPs) capable of selectively detecting USP18 activity over other ISG15 cross-reactive deubiquitinases (DUBs) by incorporating unnatural amino acids into the C-terminal tail of ISG15. Combining with a ubiquitin-based DUB ABP, the selective USP18 ABP is employed in a chemoproteomic screening platform to identify and assess inhibitors of DUBs including USP18. We further demonstrate that USP18 ABPs can be utilized to profile differential activities of USP18 in lung cancer cell lines, providing a strategy that will help define the activity-related landscape of USP18 in different disease states and unravel important (de)ISGylation-dependent biological processes.

molecular biology↗

bioRxiv2019

Chromatin interactome mapping at 139 independent breast cancer risk signals

Genome-wide association studies have identified 196 high confidence independent signals associated with breast cancer susceptibility. Variants within these signals frequently fall in distal regulatory DNA elements that control gene expression. We designed a Capture Hi-C array to enrich for chromatin interactions between the credible causal variants and target genes in six human mammary epithelial and breast cancer cell lines. We show that interacting regions are enriched for open chromatin, histone marks for active enhancers and transcription factors relevant to breast biology. We exploit this comprehensive resource to identify candidate target genes at 139 independent breast cancer risk signals, and explore the functional mechanism underlying altered risk at the 12q24 risk region. Our results demonstrate the power of combining genetics, computational genomics and molecular studies to rationalize the identification of key variants and candidate target genes at breast cancer GWAS signals.

molecular biology↗

bioRxiv2019

Translation and Transforming Activity of a Circular RNA from Human Papillomavirus

Bioinformatics and in vitro studies have revealed that single-stranded circular RNAs (circRNAs), generated through backsplicing, occur more extensively than initially appreciated. While the functions of most circRNAs are unknown, binding of microRNAs (miRNA), regulation of splicing and transcription, and translation into proteins have all been demonstrated for specific circRNAs. Virally-derived circRNAs have recently been described in gamma-herpesviruses. Here, we report that oncogenic human papillomaviruses (HPV) generate circRNAs, including ones which encompass the entire coding region of the E7 oncogene (circE7). HPV16 circE7 can be detected by both inverse RT-PCR and Northern blots of HPV16-transformed cell lines. CircE7 is N6-methyladenosine (m6A) modified, preferentially localized to the cytoplasm, and can be translated to produce E7 oncoprotein. Specific disruption of circE7 in CaSki cervical carcinoma cells decreased E7 protein levels, inhibited cell proliferation, and inhibited the ability of the cells to form colonies in soft agar. Analysis of TCGA RNA-seq data demonstrates that HPV-positive cancers have abundant circE7 RNAs. These results provide evidence that virally-derived, protein-encoding circular RNAs have biologically important functions with relevance to the transforming properties of HPV.

molecular biology↗

bioRxiv2019

DNA energy constraints shape biological evolutionary trajectories

Most living systems rely on double-stranded DNA (dsDNA) to store their genetic information and perpetrate themselves. Thus, the biological information contained within a dsDNA molecule, in terms of a linear sequence of nucleotides, has been considered the main target of the evolution. However, in this information-centred perspective, certain DNA sequence symmetries are difficult to explain. Here we show that these patterns can emerge from the physical peculiarities of the dsDNA molecule itself and the maximum entropy principle alone, rather than from biological or environmental evolutionary pressure. Our predictions are valid for both prokaryotes and eukaryotes, and also inform the interpretation of observed codon biases and context-dependent mutation patterns in human populations. Our results suggest that the double helix energy constraints and, more generally, the physical properties of the dsDNA are the hard drivers of the overall DNA sequence architecture, whereas the biological selective processes act as soft drivers, which only under extraordinary circumstances overtake the overall entropy content of the genome.

molecular biology↗

bioRxiv2026

Cysteine Glutathionylation as a Global Dynamic Regulator of Protein Active Site Accessibility and Protein Complex Formation

Protein-glutathionylation is traditionally viewed as a protective mechanism that shields cysteine-residues from irreversible oxidative damage. Its broader functional roles remain poorly understood, in part due to technical limitations in detecting this modification at scale. Here, we develop and leverage a new mass-spectrometry approach that preserves protein-glutathionylation, thereby revealing its widespread distribution across the proteome in cell models, worms, mice and human cardiac tissues. In all cases, we find that glutathionylation sites are enriched at both protein-protein interfaces and protein-active sites, and are highly conserved across species. We find that glutathionylation is dynamically redistributed in response to environmental challenges, thereby driving remodelling of cellular protein-protein interaction (PPI) networks, and access to protein active sites, with functional and phenotypic consequences. Finally, we show that glutathionylation accumulates on key cardiac-sarcomeric proteins in aged-mice and human cardiomyopathy biopsies, revealing its role in cardiovascular dysfunction. These findings reposition glutathionylation as a crucial regulatory PTM, akin to phosphorylation, that orchestrates adaptive cellular responses. This work redefines the role of glutathionylation, with broad implications for cell biology and disease.

molecular biology↗

bioRxiv2026

Telomere Length as an Indicator of Lifestyle-Related Biological Aging in Hypertensive Adults: A Pilot Exploratory Study

BackgroundMultiple epidemiological studies have given a global perspective that hypertension leads to changes in telomere length (TL). This study aimed to investigate the association between leukocyte telomere length and lifestyle factors among individuals with hypertension, a family history of the disease and healthy controls. MethodsThis pilot exploratory cross-sectional study included 45 participants (n = 15 per group) divided into hypertensive, familial hypertensive, and control groups. Lifestyle behaviors were assessed using the FANTASTIC Lifestyle Checklist, evaluating domains such as physical activity, diet, sleep, and stress. Relative telomere length (T/S ratio) of leucocytes was measured from peripheral blood samples using quantitative real-time PCR (qPCR). Data were analyzed using SPSS software, with ANOVA, Kruskal-Wallis test, and multivariable linear regression applied for statistical analysis. ResultsRelative telomere length differed significantly among the study groups (p = 0.008), with hypertensive participants demonstrating the highest median telomere-to-single-copy gene (T/S) ratio (2.66), followed by familial hypertensive (1.25) and control participants (0.87). Total lifestyle scores also varied significantly across groups (p < 0.001), with hypertensive individuals exhibiting higher mean scores (77.07 {+/-} 5.59) than familial hypertensive (67.20 {+/-} 3.00) and control participants (67.07 {+/-} 5.97). A modest positive correlation was observed between total lifestyle score and relative telomere length (r = 0.329, p < 0.05), suggesting that healthier lifestyle behaviors in diagnosed hypertensive subjects following healthy lifestyles and regular medications were associated with longer telomeres. However, in multivariable linear regression analyses, lifestyle score, age, gender, hypertension status, and family history of hypertension were not independently associated with telomere length (all p > 0.05). ConclusionLifestyle scores were positively associated with telomeres, and significantly higher telomere ratios in hypertensive subjects suggest complex biological interactions that require further investigation through larger longitudinal studies in the target population.

Molecular Biology↗

bioRxiv2026

Multidimensional atlas of RNA-regulated proteins revealed by RNA-dependent thermal proteome profiling

RNA and proteins interact pervasively in cellular processes, yet the functional relevance of most RNA-binding proteins remains unknown. To address this, we developed RNA-dependent thermal proteome profiling (RTPP), a method that identifies RNA-regulated proteins (RRPs) by detecting changes in a proteins structural stability upon RNA binding, without requiring crosslinking or enrichment. Applying RTPP in HEK293T cells revealed 1,664 RRPs, including 257 previously undetected RNA-binding "orphans". One such orphan, SGK3, binds the lncRNA CASC15, facilitating its PtdIns(3)P binding, endosomal recruitment and kinase activity. We further generated a tissue-specific RRP atlas, identifying interactions unique to particular organs, including hippocampus-specific RRPs dysregulated in Alzheimers disease. Integrating RTPP with proximity labeling resolved RNA-binding heterogeneities across subcellular compartments. Live-cell RNase treatment also uncovered proteins associated with cell surface RNA, revealing that RO60 form nanoscale clusters with glycoRNA. RTPP provides a powerful approach to decipher functional RNA-protein interactions across multiple biological dimensions.

molecular biology↗