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bioRxiv2021

A BioID-derived proximity interactome for SARS-CoV-2 proteins

The novel coronavirus SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic and has caused a major health and economic burden worldwide. Understanding how SARS-CoV-2 viral proteins behave in host cells can reveal underlying mechanisms of pathogenesis and assist in development of antiviral therapies. Here we use BioID to map the SARS-CoV-2 virus-host interactome using human lung cancer derived A549 cells expressing individual SARS-CoV-2 viral proteins. Functional enrichment analyses revealed previously reported and unreported cellular pathways that are in association with SARS-CoV-2 proteins. We have also established a website to host the proteomic data to allow for public access and continued analysis of host-viral protein associations and whole-cell proteomes of cells expressing the viral-BioID fusion proteins. Collectively, these studies provide a valuable resource to potentially uncover novel SARS-CoV-2 biology and inform development of antivirals.

molecular biology↗

bioRxiv2023

The AccelerAge framework: A new statistical approach to predict biological age based on time-to-event data

Aging is a multifaceted and intricate physiological process characterized by a gradual decline in functional capacity, leading to increased susceptibility to diseases and mortality. While chronological age serves as a strong risk factor for age-related health conditions, considerable heterogeneity exists in the aging trajectories of individuals, suggesting that biological age may provide a more nuanced understanding of the aging process. However, the concept of biological age lacks a clear operationalization, leading to the development of various biological age predictors without a solid statistical foundation. This paper addresses these limitations by proposing a comprehensive operationalization of biological age, introducing the "AccelerAge" framework for predicting biological age, and introducing previously underutilized evaluation measures for assessing the performance of biological age predictors. The AccelerAge framework, based on Accelerated Failure Time (AFT) models, directly models the effect of candidate predictors of aging on an individuals survival time, aligning with the prevalent metaphor of aging as a clock. We compare predictors based on the AccelerAge framework to a predictor based on the GrimAge predictor, which is considered one of the best-performing biological age predictors, using simulated data as well as data from the UK Biobank and the Leiden Longevity Study. Our approach seeks to establish a robust statistical foundation for biological age clocks, enabling a more accurate and interpretable assessment of an individuals aging status.

molecular biology↗

bioRxiv2024

Ribonuclease activity undermines immune sensing of naked extracellular RNA

The plasma membrane and the membrane of endosomal vesicles are considered physical barriers preventing extracellular RNA uptake. While naked RNA can be spontaneously internalized by certain cells types, functional delivery of naked RNA into the cytosol has been rarely observed. Here we show that extracellular ribonucleases, mainly derived from cell culture supplements, have so far hindered the study of extracellular RNA functionality. In the presence of active ribonuclease inhibitors (RI), naked bacterial RNA is pro-inflammatory when spiked in the media of dendritic cells and macrophages. In murine cells, this response mainly depends on the action of endosomal Toll-like receptors. However, we also show that naked RNA can perform endosomal escape and engage with cytosolic RNA sensors and ribosomes. For example, naked mRNAs encoding reporter proteins can be spontaneously internalized and translated by a variety of cell types, in an RI-dependent manner. In vivo, RI co-injection enhances the activation induced by naked extracellular RNA on splenic lymphocytes and myeloid-derived leukocytes. Furthermore, naked extracellular RNA is inherently pro-inflammatory in ribonuclease-poor compartments such as the peritoneal cavity. Overall, these results demonstrate that naked RNA is bioactive and does not need encapsulation inside synthetic or biological lipid vesicles for functional uptake, making a case for nonvesicular extracellular RNA-mediated intercellular communication.

molecular biology↗

bioRxiv2025

Extended nuclear glycosylation is a common post-translational modification

In eukaryotes, glycans modify proteins in the secretory pathway and the extracellular space. Aside from nucleocytoplasmic O-GlcNAc, glycosylation is not considered a relevant post-translational modification in other cellular compartments. Here, we challenge this long-standing paradigm by showing that extended O-glycans are commonly found on intranuclear proteins. Through comprehensive genetic and biochemical analyses, we conclusively demonstrate that these O-glycans stem from the secretory pathway, yet are found on nuclear proteins across mammalian cell lines and primary cells. Using knock-out cell lines, we show mechanistically that nuclear glycans are shuttled to the nucleus via active vesicular transport. We identify several of these intranuclear glycoproteins as RNA-binding proteins, including KHSRP/FUBP2, RBM12, and RPP30. Lastly, we show that site-specific glycosylation of RPP30 is crucial for effective tRNA processing. Overall, our findings suggest a much broader role for glycosylation in regulating cellular functions and open up investigation into the role of glycans in more biological processes.

molecular biology↗

bioRxiv2026

Vitamin B12 Improves Skeletal Muscle Mitochondrial Biology in Aged Mice

Age-related skeletal muscle deterioration is a commonly reported disability among older adults, attributed to several factors including mitochondrial dysfunction, a major hallmark of aging. Therapies to attenuate or reverse mitochondrial decline are limited. Despite identified positive relationships between vitamin B12 (B12) and mitochondrial biology, the impact of B12 supplementation on skeletal muscle mitochondria, in advanced aged, has not been examined. Thus, the impact of B12 supplementation on skeletal muscle mitochondrial biology was examined in (i) aged female mice, given 12 weeks of B12 supplementation (SUPP) or vehicle control, and (ii) in human primary myotubes. In the mouse model, mitochondrial DNA and content were measured with PCR and citrate synthase activity, respectively; mitochondrial morphology was examined using transmission electron microscopy; mitochondrial function was examined using extracellular metabolic flux analysis; and proteins and pathway enrichment was identified with proteomics. In the cell model, ROS and glutathione was measured using luminescent assays. The results demonstrated that SUPP in aged mice increased muscle mitochondrial content and improved morphology. Further, differentially expressed proteins were enriched in TCA cycle, OXPHOS, and oxidative stress pathways. In the cell model, B12 supplementation reduced ROS levels. This is the first study, to our knowledge, examining the impact of B12 supplementation on skeletal muscle mitochondrial biology in aged female mice. Results suggest that B12 supplementation improves mitochondrial biology in aged female mice.

molecular biology↗

bioRxiv2021

Cohesin Releasing Factor WAPL Regulates Genome Structure and Function of Mature T Cells

The cohesin complex modulates gene expression and cellular functions by shaping three-dimensional (3D) organization of chromatin. WAPL, cohesins DNA release factor, regulates 3D chromatin architecture. The 3D genome structure and its relevance to mature T cell functions in vivo is not well understood. We show that in vivo lymphopenic expansion, and allo-antigen driven proliferation, alters the 3D structure and function of the genome in mature T cells. Conditional deletion of Wapl in T cells reduced long-range genomic interactions, altered chromatin A/B compartments and interactions within topologically associating domains (TADs) of the chromatin in T cells at baseline. Comparison of chromatin structure in normal and WAPL-deficient T cells after lymphopenic and allo-antigen driven stimulation revealed reduced loop extensions with changes in cell cycling genes. WAPL-mediated changes in 3D architecture of chromatin regulated activation, cycling and proliferation of T cells in vitro and in vivo. Finally, WAPL-deficient T cells demonstrated reduced severity of graft-versus-host disease (GVHD) following experimental allogeneic hematopoietic stem cell transplantation. These data collectively characterize 3D genomic architecture of T cells in vivo and demonstrate biological and clinical implications for its disruption by cohesin release factor WAPL.

molecular biology↗

bioRxiv2024

Milk Fat Globule Membrane-Containing Protein Powder Promotes Fitness in Caenorhabditis elegans

Milk-derived peptides and milk fat globule membrane (MFGM) have gained interest as health-promoting food ingredients. However, the mechanisms by which these nutraceuticals modulate the function of biological systems often remain unclear. We utilized Caenorhabditis elegans to elucidate how milk-derived Protein powders rich in MFGM, previously used in a clinical trial, affect the physiology of this model organism. Our results demonstrate that Protein powders do not affect lifespan but promote the fitness of the animals. Surprisingly, gene expression analysis revealed that Protein powders decrease the expression of genes functioning on innate immunity, which also translates into reduced survival on pathogenic bacteria. One of the innate immunity-associated genes showing reduced expression upon Protein powder supplementation is cpr-3, the homolog of human cathepsin B. Interestingly, knockdown of cpr-3 enhances fitness, but not in Protein powder-treated animals, suggesting that protein powders contribute to fitness by downregulating the expression of this gene. In summary, this research highlights the value of C. elegans in testing the biological activity of food supplements and nutraceuticals. Furthermore, this study should encourage investigations into whether milk-derived peptides and MFGM mediate their beneficial effects through the modulation of cathepsin B expression in humans.

molecular biology↗

bioRxiv2025

Development of Motif-Specific Monoclonal Antibodies for Global Protein Citrullination Detection with Minimal Cross-Reactivity to Homocitrullination

Protein citrullination, a post-translational modification (PTM) catalyzed by peptidylarginine deiminases (PADs), plays critical roles in biological processes such as immunity, gene regulation, and inflammation. Dysregulated citrullination is implicated in diseases including rheumatoid arthritis, multiple sclerosis, and cancer, making it a potential biomarker and therapeutic target. Immunodetection is the most commonly used technique to study citrullination. However, most commercially available antibodies against citrullination are either protein-specific or lack the sensitivity and specificity of the broad variety of modified proteins. In addition, existing anti-pan citrullination antibodies often fail to distinguish citrullination from homocitrullination, a chemically similar modification on lysine. This cross-reactivity limits their utility in deciphering the distinct biological roles of these PTMs. To address these challenges, we employed a motif-based strategy to generate monoclonal antibodies against citrullination. This approach leverages PAD enzyme sequence preferences from human proteome, enhancing specificity for citrullination while minimizing cross-reactivity with homocitrullination. We immunized rats with a pool of over 490,000 citrullinated peptides, designed to represent common citrullination motifs in human tissue proteomes. Two monoclonal antibody clones were established and validated for sensitivity and specificity using ELISA and western blot against in vitro citrullinated and homocitrullinated proteomes, as well as ionomycin-activated human neutrophils. Both clones demonstrated great sensitivity to diverse citrullinated proteins, robust discrimination against homocitrullination, and quantitative readout in biological samples. These novel antibodies provide powerful tools for studying global citrullination dynamics and hold promise for biomarker discovery and diagnostic applications in diseases involving PAD dysregulation.

molecular biology↗

bioRxiv2025

The CAGE complex: a hollow, megadalton, protein assembly in prokaryotic and eukaryotic microbes

We report the discovery and structure of a previously unknown [~]1 MDa hollow protein assembly, identified during a survey of ciliary complexes from the ciliate Tetrahymena thermophila. By combining mass spectrometry, structure prediction, and cryo-electron microscopy, we define a homotetrameric cage-like complex with a distinctive elliptical architecture and a large internal cavity. A sequence survey revealed several thousand homologs spanning diverse unicellular eukaryotes--including green algae, fungi, amoebozoans, choanoflagellates, and SAR lineages--as well as predominantly gram-negative bacteria, indicating an ancient evolutionary origin and arguing against a eukaryote-specific function. We determined a near-atomic resolution structure of the complex from the slime mold Dictyostelium discoideum, demonstrating conservation of overall architecture and cavity despite low sequence identity. Together, these results establish the CAGE complex (Conserved Assembly in Gram-negative bacteria and Eukaryotes) as a new class of large protein cage broadly distributed across the tree of life. While its biological function remains unknown, its size, architecture, and conservation suggest possible roles in transport or protein/RNA homeostasis.

molecular biology↗

bioRxiv2025

Liquid-Liquid Phase Separation-mediated formation of amyloid fibrils from DcpS scavenger enzymes.

Decapping Scavenger (DcpS) enzyme was initially identified by its ability to hydrolyze the cap structure resulting from mRNA decay. Human DcpS is an established target for acute myeloid leukemia (AML) and hepatic metastasis. Recently, the protein has been linked to neuronal development regulation and implicated in certain developmental neurological disorders. Here we demonstrate for the first time that DcpS undergoes misfolding in vitro, leading to the formation of amyloid-like fibrils. Fibrillization was observed for human and nematode (C. elegans) DcpS using transmission electron microscope (TEM) imaging, Thioflavin T (ThT) fluorescence assay, Fourier-transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy, differential scanning fluorimetry (DSF), and dynamic light scattering (DLS). Additionally, the DcpSINS15 insertional mutant linked to the Al-Raquad syndrome, exhibited accelerated fibril aggregation kinetics compared to the wild type protein. Moreover, we show that the DcpS species investigated in this study undergo liquid-liquid phase separation (LLPS) prior to amyloid-formation. We propose that the LLPS phase transition underlies the intricate kinetics (e.g. lack of a clearly-resolved lag phase) of the misfolding process. As the physiological implications of the here-reported propensity of DcpS to lose its biological function through the coupled LLPS-fibrillization transition remain to be elucidated, this work lays the groundwork for further studies on this phenomenon.

molecular biology↗

bioRxiv2025

African Green Monkeys Respond to Synthetic AB Oligomers with Persistent Alzheimers-like Activation

Wild African green monkeys (AGMs) provide a promising alternative to congenic rodent models because of their closer evolutionary relationship to humans and natural genetic variation. They share key physiological and biochemical traits with humans, including lifespan, neuroanatomy, vascular structure, and inflammatory responses. Unlike rodents, AGMs naturally develop Alzheimers-like amyloid-{beta} (A{beta}) plaques and tau tangles with age. Immunohistochemical studies further show that AGMs inoculated with synthetic A{beta} oligomers (A{beta}O) exhibit hyperphosphorylated tau and neuroinflammation one year later, in the absence of overt neurodegeneration. The AGM body size permits collection of cerebrospinal fluid (CSF) and CSF derived extracellular vesicles (EV) from living individuals, which are key sources of Alzheimers disease biomarkers that can be monitored during disease progression. Here, we evaluate A{beta}O treated AGMs at the systems level using proteomics of CSF and phosphatidylserine affinity isolated EVs (EVps). We optimized a workflow to obtain paired CSF and EVps proteomics from <1 mL volumes, i.e. comparable to human liquid biopsy. Our measurements reveal robust, persistent AD-like responses at the biochemical level without overt loss of cognitive function. As such, these findings in AGMs suggest potential alternatives for disease tracking or point to protective mechanisms for limiting disease progression in AD. HighlightsO_LIDual proteomics of African green monkeys transiently challenged with synthetic A{beta} oligomers (A{beta}O) C_LIO_LIPhosphotidylserine (TIM4) based workflow enables CSF and EV profiling using clinical volumes C_LIO_LIOne year post-A{beta}O: vascular-inflammatory pathways rise; neuronal-axonal pathways fall C_LIO_LIA{beta}Os drive human-AD-like proteome shifts on time scales shorter than cognitive decline C_LI In briefWild African green monkeys (AGMs) offer a translational model for early Alzheimers biology, with physiology more similar to humans. We transiently exposed AGMs to synthetic A{beta} oligomers and, 12 months later, profiled paired proteomes from whole CSF and a CSF subcompartment enriched for extracellular vesicles. Despite no overt cognitive decline, AGM proteomes showed persistent Alzheimers-like remodeling, particularly in vascular, inflammatory, and neuronal systems. Parallel analysis of the CSF subcompartment revealed proteins and pathways under-represented in bulk CSF, sharpening disease-relevant signals and candidate biomarkers. This systems-level, longitudinal study establishes AGMs as a powerful platform for liquid biopsy discovery and illuminates basic biology of molecular responses to soluble A{beta} oligomers accompany and potentially protect against neurodegeneration.

systems biology↗

bioRxiv2023

MicroRNA-423-5p Mediates Cocaine-Induced Smooth Muscle Cell Contraction by Targeting Cacna2d2

BackgroundCocaine abuse increases the risk of atherosclerotic cardiovascular disease (CVD) and causes acute coronary syndromes (ACS) and hypertension (HTN). Significant research has explored the role of the sympathetic nervous system mediating the cocaine effects on the cardiovascular (CV) system. However, the response of the sympathetic nervous system alone is insufficient to completely account for the CV consequences seen in cocaine users. Here, we examined the role of microRNAs (miRNAs) in mediating the effect of cocaine on the CV system. MiRNAs regulate many important biological processes and have been associated with both response to cocaine and CV disease development. Multiple miRNAs have altered expression in the CV system (CVS) upon cocaine exposure. Herein, we examined the role of microRNA-423-5p and the downstream signaling events in regulating cocaine-induced mouse aortic smooth muscle cell (SMC) contraction. MethodsTo understand the molecular mechanisms underlying the cocaine response in the CV system, we studied the role of miRNA-423-5p and its target Cacna2d2 in the regulation of intracellular calcium concentration and SMC contractility, a critical factor in the modulation of blood pressure (BP). We used in vivo models to evaluate BP and aortic stiffness. ResultsIn vitro, Cocaine treatment decreased miR-423-5p expression and increased Cacna2d2 expression, which led to elevated intracellular calcium concentrations and increased SMC contractility. Overexpression of miR-423-5p, silencing of its target Cacna2d2, and treatment with a calcium channel blocker reversed the elevated SMC contractility caused by cocaine. In contrast, suppression of miR-423-5p increased the intracellular calcium concentration and SMC contractibility. In vivo, overexpression of miR-423-5p ameliorated the increase in BP and aortic stiffness associated with cocaine use. ConclusionsMiR-423-5p regulates SMC contraction by modulating Cacna2d2 expression increasing intracellular calcium concentrations. Modulation of miR-423-5p--Cacna2d2--Calcium transport pathway may represent a novel therapeutic strategy to improve cocaine-induced hypertension and aortic stiffness.

molecular biology↗

bioRxiv2018

Bayesian inference and comparison of stochastic transcription elongation models

Transcription elongation can be modelled as a three step process, involving polymerase translocation, NTP binding, and nucleotide incorporation into the nascent mRNA. This cycle of events can be simulated at the single-molecule level as a continuous-time Markov process using parameters derived from single-molecule experiments. Previously developed models differ in the way they are parameterised, and in their incorporation of partial equilibrium approximations.\n\nWe have formulated a hierarchical network comprised of 12 sequence-dependent transcription elongation models. The simplest model has two parameters and assumes that both translocation and NTP binding can be modelled as equilibrium processes. The most complex model has six parameters makes no partial equilibrium assumptions. We systematically compared the ability of these models to explain published force-velocity data, using approximate Bayesian computation. This analysis was performed using data for the RNA polymerase complexes of E. coli, S. cerevisiae and Bacteriophage T7.\n\nOur analysis indicates that the polymerases differ significantly in their translocation rates, with the rates in T7 pol being fast compared to E. coli RNAP and S. cerevisiae pol II. Different models are applicable in different cases. We also show that all three RNA polymerases have an energetic preference for the posttranslocated state over the pretranslocated state. A Bayesian inference and model selection framework, like the one presented in this publication, should be routinely applicable to the interrogation of single-molecule datasets.\n\nAuthor summaryTranscription is a critical biological process which occurs in all living organisms. It involves copying the organisms genetic material into messenger RNA (mRNA) which directs protein synthesis on the ribosome. Transcription is performed by RNA polymerases which have been extensively studied using both ensemble and single-molecule techniques (see reviews: [1, 2]). Single-molecule data provides unique insights into the molecular behaviour of RNA polymerases. Transcription at the single-molecule level can be computationally simulated as a continuous-time Markov process and the model outputs compared with experimental data. In this study we use Bayesian techniques to perform a systematic comparison of 12 stochastic models of transcriptional elongation. We demonstrate how equilibrium approximations can strengthen or weaken the model, and show how Bayesian techniques can identify necessary or unnecessary model parameters. We describe a framework to a) simulate, b) perform inference on, and c) compare models of transcription elongation.

molecular biology↗

bioRxiv2019

Identification of flowering-time genes in mast flowering plants using de novo transcriptomic analysis

Mast flowering is synchronised highly variable flowering by a population of perennial plants over a wide geographical area. High seeding years are seen as a threat to native and endangered species due to high predator density caused by the abundance of seed. An understanding of the molecular pathways that influence masting behaviour in plants could provide better prediction of a forthcoming masting season and enable conservation strategies to be deployed. In this study, a high-throughput large-scale RNA-sequencing was performed on two masting plant species, Celmisia lyallii (Asteraceae), and Chionochloa pallens (Poaceae) to develop a reference transcriptome for functional and molecular analysis. An average total of 33 million 150 base-paired reads, for both species, were assembled using the Trinity pipeline, resulting in 151,803 and 348,649 transcripts respectively for Celmisia and Chionochloa. The two datasets generated were blasted against the publicly available databases, TAIR, Swiss-Prot, non-redundant protein (nr), KEGG and COG for unigene annotations. On average, 56% of the unigenes were finally annotated with gene descriptions mapped to known protein sequences for both the species. Gene ontology analysis was then performed on the assembled reference transcriptomes, categorising the transcripts on the basis of putative biological processes, molecular function, and cellular localisation. A total of 543 transcripts from Celmisia and 470 transcripts from Chionochloa were also mapped to unique flowering-time proteins identified in Arabidopsis, suggesting the conservation of the flowering network in these wild alpine plants, growing in natural field conditions. These genes can further be analysed to understand the molecular regulation of the reproductive phase transition in the masting plants.

plant biology↗

bioRxiv2022

Unexpected growth of a classic yeast auxotroph

Organisms must either synthesize or assimilate essential organic compounds to survive. The homocysteine synthase Met15 has been considered essential for inorganic sulfur assimilation in yeast since its discovery in the 1970s. As a result, MET15 has served as a genetic marker for hundreds of experiments that play a foundational role in eukaryote genetics and systems biology. Nevertheless, we demonstrate here through structural and evolutionary modeling, in vitro kinetic assays, and genetic complementation, that an alternative homocysteine synthase encoded by the previously uncharacterized gene YLL058W enables cells lacking Met15 to assimilate enough inorganic sulfur for survival and proliferation. These cells however fail to grow in patches or liquid cultures unless provided with exogenous methionine or other organosulfurs. We show that this growth failure, which has historically justified the status of MET15 as a classic auxotrophic marker, is largely explained by toxic accumulation of the gas hydrogen sulfide due to a metabolic bottleneck. When patched or cultured with a hydrogen sulfide chelator, and when propagated as colony grids, cells without Met15 assimilate inorganic sulfur and grow, and cells with Met15 achieve even higher yields. Thus, Met15 is not essential for inorganic sulfur assimilation in yeast. Instead, MET15 is the first example of a yeast gene whose loss conditionally prevents growth in a manner that depends on local gas exchange. Our results have broad implications for investigations of sulfur metabolism, including studies of stress response, methionine restriction, and aging. More generally, our findings illustrate how unappreciated experimental variables can obfuscate biological discovery.

molecular biology↗

bioRxiv2022

Core and Flanking bHLH-PAS:DNA interactions mediate specificity and drive obesity

The basic-Helix-Loop-Helix Per-Arnt-Sim (PAS) homology domain (bHLH-PAS) transcription factor (TF) family comprises critical biological sensors of physiological (hypoxia, tryptophan metabolites, neuronal activity, and appetite) and environmental (diet derived metabolites and environmental pollutants) stimuli to regulate genes involved in signal adaptation and homeostasis1. bHLH TFs bind DNA as homo or heterodimers via E-box (CANNTG) response elements, however the DNA binding specificity of the PAS domain-containing bHLH subfamily remains unresolved1. We systematically analysed cognate DNA binding hierarchies of prototypical bHLH-PAS family members (ARNT, ARNT2, HIF1, HIF2, AhR, NPAS4, SIM1) and demonstrate distinct core (NNCGTG) specificities for different heterodimer classes. The results also show that bHLH-PAS TFs bind over a large footprint 12-15bp and recognise preferential DNA sequences flanking the core. For example, specificity beyond otherwise identical core binding by SIM1 and the HIFs is mediated through N-terminal HIF-DNA interactions. We also reveal an intimate relationship between DNA shape and both core and flanking TF binding allowing motif sequence flexibility and underpinning TF binding specificity. Furthermore, DNA-shape affinity relationships revealed that novel downstream PAS-A-loop DNA interactions are associated with AT-rich sequences that lead to high-affinity binding, and that loss of this function underpins a monogenic cause of human hyperphagic obesity in a recapitulated SIM1.R171H knock-in mouse model. Importantly, models of protein-DNA binding accurately predict in vivo occupancy, while response element methylation blocks DNA binding and predicts cell type specific chromatin occupancy. These data provide a definitive and accurate map of bHLH-PAS TF specificity and target selectivity through novel flanking protein-DNA interactions that are crucial for in vivo biological function.

molecular biology↗

bioRxiv2022

Proteomic Analysis Defines the Interactome of Telomerase in the Protozoan Parasite, Trypanosoma brucei

Telomerase is a ribonucleoprotein enzyme responsible for maintaining the telomeric end of the chromosome. The telomerase enzyme requires two main components to function: the telomerase reverse transcriptase (TERT) and the telomerase RNA (TR), which provides the template for telomeric DNA synthesis. TR is a long noncoding RNA, which forms the basis of a large structural scaffold upon which many accessory proteins can bind and form the complete telomerase holoenzyme. These accessory protein interactions are required for telomerase activity and regulation inside cells. The interacting partners of TERT have been well studied in yeast, human, and Tetrahymena models, but not in lower eukaryotes, including clinically relevant human parasites. Here, using the protozoan parasite, Trypanosoma brucei (T. brucei) as a model, we have identified the interactome of T. brucei TERT (TbTERT) using a mass spectrometry-based approach. We identified previously known and unknown interacting factors of TbTERT, highlighting unique features of T. brucei telomerase biology. These unique interactions with TbTERT, suggest mechanistic differences in telomere maintenance between T. brucei and other eukaryotes.

molecular biology↗

bioRxiv2023

Human telomere length is chromosome specific and conserved across individuals

Short telomeres cause age-related disease and long telomeres predispose to cancer; however, the mechanisms regulating telomere length are unclear. To probe these mechanisms, we developed a nanopore sequencing method, Telomere Profiling, that is easy to implement, precise, and cost effective with broad applications in research and the clinic. We sequenced telomeres from individuals with short telomere syndromes and found similar telomere lengths to the clinical FlowFISH assay. We mapped telomere reads to specific chromosome end and identified both chromosome end-specific and haplotype-specific telomere length distributions. In the T2T HG002 genome, where the average telomere length is 5kb, we found a remarkable 6kb difference in lengths between some telomeres. Further, we found that specific chromosome ends were consistently shorter or longer than the average length across 147 individuals. The presence of conserved chromosome end-specific telomere lengths suggests there are new paradigms in telomere biology that are yet to be explored. Understanding the mechanisms regulating length will allow deeper insights into telomere biology that can lead to new approaches to disease.

molecular biology↗