Biology subjects

Miska, E. A.

Publications and source records attributed to Miska, E. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Mesenchymal stem cell-extracellular vesicles deliver microRNAs that prevent nerve growth factor-induced sensory neuron sensitization

Osteoarthritis (OA) affects 600 million individuals globally, pain being a hallmark symptom. Emerging clinical evidence supports the use of mesenchymal stem cells (MSCs) and their extracellular vesicles (MSC-EVs) for pain relief in knee OA. In mice, MSC-EVs ameliorate OA-induced pain and normalize knee-innervating neuron excitability. Moreover, it has been shown that overnight incubation of sensory neurons with MSC-EVs prevents the OA-associated mediator nerve growth factor (NGF) sensitizing sensory neurons. Here, we found that protease-mediated MSC-EV shaving inhibited MSC-EV internalization into sensory neurons and the ability of MSC-EVs to prevent NGF-induced sensitization. In addition, acute, 10-minute, exposure of sensory neurons to MSC-EVs was also insufficient to counteract NGF. We hypothesized that MSC-EVs trigger transcriptional changes and found that inhibiting transcription prevented NGF-induced sensitization. MicroRNAs (miRNAs) can be delivered to cells by MSC-EVs, and certain miRNAs regulate transcription and pain; small RNA-sequencing of our MSC-EVs identified three candidate miRNAs, miR-21-5p, miR-148a-3p and miR-451a. Using gold nanoparticle delivery, each miRNA was able to prevent NGF sensitization of sensory neurons, a combination of all three showing the most pronounced effect. These findings demonstrate that MSC-EVs prevent NGF-induced sensory neuron sensitization via cellular uptake and transcriptional regulation that is mediated by miRNAs.

neuroscience↗

bioRxiv2025

Compensatory tRNA Modification by DUS3L Confers Resistance to METTL1 Loss in Oesophageal Cancer

tRNA N-methylguanosine (mG46), installed by the METTL1/WDR4 methyltransferase, stabilizes the tRNA pool supporting high proliferative rates. Accordingly, many cancer cell lines are vulnerable to METTL1 loss, underscoring its potential as a therapeutic target. We identify a subset of cell lines insensitive to METTL1 depletion and use oesophageal lines as a model to explore the mechanism. The METTL1-resistant adenocarcinoma line OE33 preserves tRNA balance upon mG46 deficiency by elevating dihydrouridylation at position 47 (D47). Depletion of DUS-L enzymes that catalyse D16/D17/D20/D47 on tRNAs sensitises OE33 to METTL1 loss. Concomitant loss of dihydrouridylation and mG46 destabilizes the canonical L-shaped tRNA structure, disrupts coaxial helix stacking and anticodon-loop presentation, and reduces cellular fitness. Notably, METTL1-sensitive cell lines lack this compensatory pathway due to the deleterious effects of increasing their intrinsically low DUS3L levels. These findings reveal a DUS-L-mediated buffering mechanism that supports tRNA homeostasis in METTL1-resistant cancers and highlight DUS3L abundance as a potential biomarker for predicting METTL1 sensitivity. Moreover, they suggest that combined targeting of DUS-L and METTL1 may provide a therapeutic strategy for overcoming resistance in cancers, exemplified in cell lines such as OE33. HighlightsO_LIThe oesophagus cancer line OE33 shows no proliferative defect upon METTL1/WDR4 loss, despite complete lack of m7G46 on its tRNA. C_LIO_LIOE33 coordinates codon usage and abundance of cognate tRNAs in the absence of METTL1/WDR4; the METTL1/WDR4 sensitive line, OE21, fails to do so. C_LIO_LIDepleting dihydrouridine synthases (DUS-L) renders OE33 sensitive to METTL1/WDR4 loss. C_LIO_LIOE33 responds to lack of METTL1/WDR4 activity by upregulating DUS3L and its activity (D47) on tRNA; the sensitive line OE21 fails to do so. C_LIO_LIMETTL1-sensitive lines have intrinsically low DUS3L and cannot sustain higher levels. C_LIO_LIDUS3L reveals as a potential biomarker for METTL1 sensitivity. C_LI

biochemistry↗

bioRxiv2025

A SET domain-containing protein and HCF-1 maintain transgenerational epigenetic memory

Epigenetic information can be inherited through a process known as transgenerational epigenetic inheritance (TEI). TEI can be initiated and maintained by small RNAs and histone modifications. The latter includes histone methylation, deposited by proteins with methyltransferase activity, including SET domain-containing proteins. Other SET domain-containing proteins with no catalytic methyltransferase activity also adopt roles in chromatin and gene expression regulation. Here, we describe SET-24, a SET domain-containing protein in Caenorhabditis elegans that belongs to a family of catalytically inactive SET proteins. SET-24 localises to germline nuclei and is required for germline immortality. Additionally, the inheritance of small RNA-driven epigenetic silencing is compromised in set-24 mutants. Using quantitative proteomics and yeast two-hybrid assays, we found that SET-24 interacts with Host Cell Factor 1 (HCF-1), a protein involved in epigenetic regulation and associated with known chromatin remodelling complexes, like COMPASS, which deposits H3K4me3. In set-24 mutants, hundreds of genes display increased H3K4me3 levels at their transcription start sites. While these changes are not matched at the transcriptional level, small RNA production is disrupted in approximately one fifth of those genes, which are normally targeted by small RNA pathways. We propose that SET-24 is a factor required to maintain epigenetic memory in the germline by maintaining a chromatin environment permissible to small RNA biogenesis over generations.

molecular biology↗

bioRxiv2024

Transposable elements drive regulatory and functional innovation of F-box genes

Protein domains of transposable elements (TEs) and viruses increase the protein diversity of host genomes by recombining with other protein domains. By screening 10 million eukaryotic proteins, we identified several domains that define multi-copy gene families and frequently co-occur with TE/viral domains. Among these, a Tc1/Mariner transposase helix-turn-helix (HTH) domain was captured by F-box genes in the Caenorhabditis genus, creating a new class of F-box genes. For specific members of this class, like fbxa-215, we found that the HTH domain is required for diverse processes including germ granule localisation, fertility, and thermotolerance. Furthermore, we provide evidence that HSF-1 mediates the transcriptional integration of fbxa-215 into the heat-shock response by binding to Helitron TEs directly upstream of the fbxa-215 locus. The interactome of HTH-bearing F-box factors suggests roles in post-translational regulation and proteostasis, consistent with established functions of F-box proteins. Based on AlphaFold2 multimer proteome-wide screens, we propose that the HTH domain may diversify the repertoire of protein substrates that F-box factors regulate post-translationally. We further demonstrate that F-box genes repeatedly and independently captured TE domains throughout eukaryotic evolution, and describe an additional instance in zebrafish. In conclusion, we identify recurrent TE domain captures by F-box genes in eukaryotes and provide insights into how these novel proteins are integrated within host gene regulatory networks.

evolutionary biology↗

bioRxiv2024

Introgression dynamics of sex-linked chromosomal inversions shape the Malawi cichlid adaptive radiation

Chromosomal inversions contribute to adaptive speciation by linking co-adapted alleles. Querying 1,375 genomes of the species-rich Malawi cichlid fish radiation, we discovered five large inversions segregating in the benthic subradiation that each suppress recombination over more than half a chromosome. Two inversions were transferred from deepwater pelagic Diplotaxodon via admixture, while the others established early in the deep benthic clade. Introgression of haplotypes from lineages inside and outside the Malawi radiation coincided with bursts of species diversification. Inversions show evidence for transient sex linkage and a striking excess of protein changing substitutions points towards selection on neuro-sensory, physiological and reproductive genes. We conclude that repeated interplay between depth adaptation and sex-specific selection on large inversions has been central to the evolution of this iconic system.

evolutionary biology↗

bioRxiv2023

comrades-OO: An Object-Oriented R Package for Comprehensive Analysis of RNA Structure Generated using RNA crosslinking experiments

SummaryRNA (Ribonucleic Acid) molecules have secondary and tertiary structures in vivo which play a crucial role in cellular processes such as the regulation of gene expression, RNA processing, and localization. The ability to investigate these structures will enhance our understanding of their function and contribute to the diagnosis and treatment of diseases caused by RNA dysregulation. However, there are no mature pipelines or packages for processing and analysing complex in vivo RNA structural data. Here, we present COMRADES Object-Oriented (comradesOO), a novel software package for the comprehensive analysis of data derived from the COMRADES (Crosslinking of Matched RNA and Deep Sequencing) method. comradesOO offers a comprehensive pipeline from raw sequencing reads to the identification of RNA structural features. It includes read processing and alignment, clustering of duplexes, data exploration, folding and comparisons of RNA structures. comradesOO also enables comparisons between conditions, the identification of inter-RNA interactions, and the incorporation of reactivity data to improve structure prediction. Availability and ImplementationcomradesOO is freely available to non-commercial users and implemented in R, with the source code and documentation accessible at [https://CRAN.R-project.org/package=comradesOO]. The software is supported on Linux, macOS, and Windows platforms. Contactjlp76@cam.ac.uk Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2023

AMPK regulates small RNA pathway prevalence to mediate soma-to-germ line communication and establish germline stem cell quiescence

It is well established that cells communicate with each other via signaling molecules and pathways. Recent work has further indicated that this transfer of information can breach the soma-to-germ line barrier, thus permitting changes in germline gene expression in response to cellular decisions made in somatic lineages. We show that during periods of extended energy stress AMPK alters small RNA biogenesis in somatic cells, which non-autonomously regulates the quiescence of germline stem cells. By combining both genetic analyses and a novel method of miRNA imaging, we show that AMPK-mediated phosphorylation acts as a molecular switch that drives the re-allocation of the key RNA endonuclease Dicer to the miRNA synthesis pathway during the dauer stage of C. elegans. By modifying Dicer and other components of the miRNA synthesis machinery, AMPK fine-tunes the production of a population of somatic miRNAs that act as a "pro-quiescence" signal to maintain germline integrity during periods of extended energy stress, thus bridging the gap between the soma and the germ line by altering small RNA homeostasis.

molecular biology↗

bioRxiv2023

U6 snRNA m6A modification is required for accurate and efficient cis- and trans-splicing of C. elegans mRNAs

pre-mRNA splicing is a critical feature of eukaryotic gene expression. Many eukaryotes use cis-splicing to remove intronic sequences from pre-mRNAs. In addition to cis-splicing, many organisms use trans-splicing to replace the 5' ends of mRNAs with a non-coding spliced-leader RNA. Both cis- and trans-splicing rely on accurately recognising splice site sequences by spliceosomal U snRNAs and associated proteins. Spliceosomal snRNAs carry multiple RNA modifications with the potential to affect different stages of pre-mRNA splicing. Here, we show that m6A modification of U6 snRNA A43 by the RNA methyltransferase METT-10 is required for accurate and efficient cis- and trans-splicing of C. elegans pre-mRNAs. The absence of U6 snRNA m6A modification primarily leads to alternative splicing at 5' splice sites. Furthermore, weaker 5' splice site recognition by the unmodified U6 snRNA A43 affects splicing at 3' splice sites. U6 snRNA m6A43 and the splicing factor SNRNP27K function to recognise an overlapping set of 5' splice sites with an adenosine at +4 position. Finally, we show that U6 snRNA m6A43 is required for efficient SL trans-splicing at weak 3' trans-splice sites. We conclude that the U6 snRNA m6A modification is important for accurate and efficient cis- and trans-splicing in C. elegans.

molecular biology↗

bioRxiv2023

Canalisation and plasticity on the developmental manifold of Caenorhabditis elegans

How do the same mechanisms that faithfully regenerate complex developmental programs in spite of environmental and genetic perturbations also permit responsiveness to environmental signals, adaptation, and genetic evolution? Using the nematode Caenorhabditis elegans as a model, we explore the phenotypic space of growth and development in various genetic and environmental contexts. Our data are growth curves and developmental parameters obtained by automated microscopy. Using these, we show that among the traits that make up the developmental space, correlations within a particular context are predictive of correlations among different contexts. Further we find that the developmental variability of this animal can be captured on a relatively low dimensional phenoptypic manifold and that on this manifold, genetic and environmental contributions to plasticity can be deconvolved independently. Our perspective offers a new way of understanding the relationship between robustness and flexibility in complex systems, suggesting that projection and concentration of dimension can naturally align these forces as complementary rather than competing.

biophysics↗

bioRxiv2023

The histone chaperone activity of SPT2 controls chromatin structure and function in Metazoa

Histone chaperones control nucleosome density and chromatin structure. In yeast, the H3-H4 chaperone Spt2 controls histone deposition at active genes but its roles in metazoan chromatin structure and organismal physiology are not known. Here we identify the Caenorhabditis elegans orthologue of SPT2 (CeSPT-2) and show that its ability to bind histones H3-H4 is important for germline development and transgenerational epigenetic gene silencing, and that spt-2 mutants display signatures of a global stress response. Genome-wide profiling showed that CeSPT-2 binds to a range of highly expressed genes, and we find that spt-2 mutants have increased chromatin accessibility at these loci. We also show that human SPT2 controls the incorporation of new H3.3 into chromatin. Our work reveals roles for SPT2 in controlling chromatin structure and function in Metazoa.

molecular biology↗

bioRxiv2022

A highly contiguous genome assembly reveals sources of genomic novelty in the symbiotic fungus Rhizophagus irregularis

The root systems of most plant species are aided by the soil foraging capacities of symbiotic Arbuscular Mycorrhizal (AM) fungi of the Glomeromycotina subphylum. Despite recent advances in our knowledge of the ecology and molecular biology of this mutualistic symbiosis, our understanding of the AM fungi genome biology is just emerging. Presented here are the most contiguous and highest-quality nuclear and mitochondrial genome assemblies of an arbuscular mycorrhizal fungus to date, achieved through Nanopore long-read DNA sequencing and Hi-C data. This haploid genome assembly of Rhizophagus irregularis, alongside short- and long-read RNA-Sequencing data, was used to produce a comprehensive annotation catalogue of gene models, repetitive elements, small RNA loci, and DNA cytosine methylome. A phylostratigraphic gene age inference framework revealed that the birth of genes associated with nutrient transporter activity and transmembrane ion transport systems predates the emergence of Glomeromycotina. While symbiotic nutrient cycling in AM fungi relies on genes that existed in ancestor lineages, a burst of Glomeromycotina-restricted genetic innovation is also detected. Analysis of the chromosomal distribution of genetic and epigenetic features highlights evolutionarily young genomic regions that produce abundant small RNAs, suggesting active RNA-based monitoring of genetic sequences surrounding recently evolved genes. This chromosome-scale view of the genome of an AM fungus genome reveals previously unexplored sources of genomic novelty in an organism evolving under an obligate symbiotic life cycle. HighlightsO_LIAssembly of 32 highly contiguous chromosomal scaffolds for R. irregularis, with 23 complete and gapless C_LIO_LIGene annotation based on short- and long-read RNA-Seq data from different developmental stages C_LIO_LIComplete annotation set including mitochondrial genes, DNA methylome, small RNAome, repetitive/transposable elements, functional annotation C_LIO_LIIdentification of a burst of lineage-restricted genetic innovation in the Glomeromycotina subphylum C_LI

genomics↗

bioRxiv2022

Loss of the E3 ubiquitin ligases UBR-5 or HECD-1 restores Caenorhabditis elegans development in the absence of SWI/SNF function

SWItch/Sucrose Non-Fermenting (SWI/SNF) complexes are a family of chromatin remodellers that are conserved across eukaryotes. Mutations in subunits of SWI/SNF cause a multitude of different developmental disorders in humans, most of which have no current treatment options. Here we identify an alanine to valine causing mutation in the SWI/SNF subunit snfc-5 (SMARCB1 in humans) that prevents embryonic lethality in C. elegans nematodes harbouring a loss-of-function mutation in the SWI/SNF subunit swsn-1 (SMARCC1/2 in humans). Furthermore, we found that the combination of this specific mutation in snfc-5 and a loss-of-function mutation in either of the E3 ubiquitin ligases ubr-5 (UBR5 in humans) or hecd-1 (HECTD1 in humans) can restore development to adulthood in swsn-1 loss-of-function mutants that otherwise die as embryos. Using these mutant models, we established a set of 335 genes that are dysregulated in SWI/SNF mutants that arrest their development embryonically but exhibit near wild-type levels of expression in the presence of suppressor mutations that prevent embryonic lethality, suggesting that SWI/SNF promotes development by regulating this specific subset of genes. In addition, we show that SWI/SNF protein levels are reduced in swsn-1; snfc-5 double mutants and partly restored to wild-type levels in swsn-1; snfc-5; ubr-5 triple mutants, consistent with a model in which UBR-5 regulates SWI/SNF levels by tagging the complex for proteasomal degradation. Our findings establish a link between two E3 ubiquitin ligases and SWI/SNF function and suggest that UBR5 and HECTD1 might be viable therapeutic targets for the many developmental disorders caused by missense mutations in SWI/SNF subunits.

developmental biology↗

bioRxiv2022

Epigenetic inheritance of gene-silencing is maintained by a self-tuning mechanism based on resource competition

Biological systems can maintain memories over long timescales, with examples including memories in the brain and immune system. It is currently unknown how functional properties of memory systems, such as memory persistence, can be established by biological circuits. To address this question, we focus on transgenerational epigenetic inheritance in C. elegans. In response to a trigger, worms silence a target gene for multiple generations, resisting strong dilution due to growth and reproduction. Silencing may also be maintained indefinitely upon selection according to silencing levels. We show that these properties imply fine-tuning of biochemical rates in which the silencing system is positioned near the transition to bistability. We demonstrate that this behavior emerges from a generic mechanism based on competition for synthesis resources, which leads to self-organization around a critical state with broad silencing timescales. The theory makes distinct predictions and offers insights into the design principles of long-term memory systems.

systems biology↗

bioRxiv2022

Genomics of cold adaptations in the Antarctic notothenioid fish radiation.

Numerous novel adaptations characterise the radiation of notothenioids, the dominant fish group in the freezing seas of the Southern Ocean. To improve understanding of the evolution of this iconic fish group, we generated and analysed new genome assemblies for 24 species covering all major subgroups of the radiation. We present a new estimate for the onset of the radiation at 10.7 million years ago, based on a time-calibrated phylogeny derived from genome-wide sequence data. We identify a two-fold variation in genome size, driven by expansion of multiple transposable element families, and use long-read sequencing data to reconstruct two evolutionarily important, highly repetitive gene family loci. First, we present the most complete reconstruction to date of the antifreeze glycoprotein gene family, whose emergence enabled survival in sub-zero temperatures, showing the expansion of the antifreeze gene locus from the ancestral to the derived state. Second, we trace the loss of haemoglobin genes in icefishes, the only vertebrates lacking functional haemoglobins, through complete reconstruction of the two haemoglobin gene clusters across notothenioid families. Finally, we show that both the haemoglobin and antifreeze genomic loci are characterised by multiple transposon expansions that may have driven the evolutionary history of these genes.

genomics↗

bioRxiv2022

Identification of putative reader proteins of 5-methylcytosine and its derivatives in Caenorhabditis elegans RNA

Methylation of carbon-5 of cytosines (m5C) is a post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. m5C has been shown to participate in mRNA transport and maintain mRNA stability through its recognition by the reader proteins ALYREF and YBX1, respectively. We recently showed that m5C is required for Caenorhabditis elegans development and fertility under heat stress. To contribute to the understanding of how m5C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers in C. elegans. Our mass spectrometry analyses reveal unique binding proteins for each of the modifications. We validate our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m5C in vitro. The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m5C and its derivatives.

biochemistry↗

bioRxiv2022

Ecological speciation promoted by divergent regulation of functional genes within African cichlid fishes

Rapid ecological speciation along depth gradients has taken place independently and repeatedly in freshwater fishes. While the extent of genomic divergence between ecomorphs is often well understood, the molecular mechanisms facilitating such rapid diversification are typically unclear. In Lake Masoko, an East African crater lake, the cichlid Astatotilapia calliptera has diverged into shallow littoral and deep benthic ecomorphs with strikingly different jaw structures within the last 1,000 years. Using genome-wide transcriptome data from jaw tissue, we explore two major regulatory transcriptional mechanisms, expression and splicing QTL variants and examine their contribution to differential gene expression underpinning functional phenotypes. We identified 7,550 genes with significant differential expression between ecomorphs, of which 4.2% were regulated by cis-regulatory expression QTLs, and 6.4% were regulated by cis-regulatory splicing QTLs. There were also strong signals of divergent selection of differentially expressed genes that showed divergent regulation from expression, splicing or both QTL variants, including genes associated with major jaw plasticity and adaptation networks, adaptive immune system response, and oxidoreductase processes. These results suggest that transcriptome plasticity and modification have important roles during early-stage ecological speciation and demonstrate the role of regulatory-variants as important targets of selection driving ecologically-relevant divergence in gene expression that is associated with adaptive diversification.

evolutionary biology↗

bioRxiv2022

oca2 targeting using CRISPR/Cas9 in the Malawi cichlid Astatotilapia calliptera

Identifying genetic loci underlying trait variation provides insights into the mechanisms of diversification, but demonstrating causality and characterising the role of genetic loci requires testing candidate gene function, often in non-model species. Here we establish CRISPR/Cas9 editing in Astatotilapia calliptera, a generalist cichlid of the remarkably diverse Lake Malawi radiation. By targeting the gene oca2 required for melanin synthesis in other vertebrate species, we show efficient editing and germline transmission. Gene edits include indels in the coding region, likely a result of non-homologous end joining, and a large deletion in the 3' UTR due to homology-directed repair. We find that oca2 knock-out A. calliptera lack melanin, which may be useful for developmental imaging in embryos and studying colour pattern formation in adults. As A. calliptera resembles the presumed generalist ancestor of the Lake Malawi cichlids radiation, establishing genome editing in this species will facilitate investigating speciation, adaptation and trait diversification in this textbook radiation.

developmental biology↗

bioRxiv2021

Structural features within the NORAD long noncoding RNA underlie efficient repression of Pumilio activity

It is increasingly appreciated that long non-coding RNAs (lncRNAs) carry out important functions in mammalian cells, but how these are encoded in their sequences and manifested in their structures remains largely unknown. Some lncRNAs bind to and modulate the availability of RNA binding proteins, but the structural principles that underlie this mode of regulation are underexplored. Here, we focused on the NORAD lncRNA, which binds Pumilio proteins and modulates their ability to repress hundreds of mRNA targets. We probed the RNA structure and long-range RNA-RNA interactions formed by NORAD inside cells, under different stressful conditions. We discovered that NORAD structure is highly modular, and consists of well-defined domains that contribute independently to NORAD function. We discovered that NORAD structure spatially clusters the Pumilio binding sites along NORAD in a manner that contributes to the de-repression of Pumilio target proteins. Following arsenite stress, the majority of NORAD structure undergoes relaxation and forms inter-molecular interactions with RNAs that are targeted to stress granules. NORAD sequence thus dictates elaborated structural domain organization that facilitates its function on multiple levels, and which helps explain the extensive evolutionary sequence conservation of NORAD regions that are not predicted to directly bind Pumilio proteins.

molecular biology↗