Biology subjects

Bonasio, R.

Publications and source records attributed to Bonasio, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Socially regulated genes are spatially hyperconnected to enhancers in the ant brain

Caste identity in Harpegnathos saltator ants remains plastic beyond development and throughout adulthood. Adult Harpegnathos workers can become dominant reproductives, known as "gamergates," through a social caste transition that involves extensive transcriptional and cellular remodeling of the brain. To gain insight into the epigenetic regulation of this process, we generated comprehensive, caste-specific epigenomic atlases of the Harpegnathos brain, including chromatin accessibility, histone modifications, and 3D genome architecture. Using these data we refined the genome assembly, annotated enhancers, and linked them to target genes. We then identified candidate 3D-architectural factors, many of which were specifically upregulated in gamergate brains. Promoters of genes upregulated during the worker-gamergate transition formed an unusually high number of 3D chromatin contacts with their regulatory regions, and most of these contacts were already present in workers. We propose that the pre-existing hyper-connectivity of socially regulated genes is essential to adult brain plasticity and behavioral reprogramming.

genomics↗

bioRxiv2025

Pseudouridine selects RNAs for extracellular transport

RNAs move through the extracellular space to transmit information between cells, including mammalian neurons, yet how specific RNAs are channeled into these extracellular routes is unknown. Using genome-wide CRISPR screening, proteomics, and high-sensitivity transcriptomics in a neuronal model system, we identify domesticated retroviral proteins and RNA-modifying enzymes that regulate RNA loading into and transportation via extracellular vesicles. We show that the pseudouridine synthase PUS1 is a key determinant of RNA trafficking, and that its catalytic product in RNA, pseudouridine, is enriched in extracellular RNAs from transformed and primary neurons. Furthermore, the presence of pseudouridine on select RNAs is both necessary and sufficient for their extracellular export. Finally, we show that myosin light chain 6 (MYL6) is a pseudouridine-binding protein required for secretion of synthetic and endogenous RNAs. These findings reveal a biochemical code linking chemical RNA modification to extracellular transport, and establish a framework to study the function of extracellular RNAs in the nervous system and beyond.

molecular biology↗

bioRxiv2025

Pooled scanning of protein variants identifies novel RNA-binding mutants

Binding to RNA has been observed for an ever-increasing number of proteins, which often have other functions. The contributions of RNA binding to protein function are best discerned by studying separation-of-function mutants that hamper interaction with RNA without affecting other aspects of protein function. To design these mutants, we need precise knowledge of the residues that contribute to the affinity of the protein for its RNA ligands. Here, we present RBR-scan: a technology to simultaneously measure RNA-binding affinity of a large number of protein variants. We fused individual variants with unique peptide barcodes optimized for detection by mass spectrometry (MS), purified protein pools from single bacterial culture, and assayed proteins in parallel for RNA binding. Mutations in the MS2 coat protein known to impair RNA-binding were correctly identified, as well as a previously unreported mutant, which we validated with orthogonal biochemical methods. We used RBR-scan to discover novel RNA-binding mutants in the cancer-associated splicing regulator SRSF2. Together, our results demonstrate that RBR-scan is a powerful and scalable platform for linking RNA-binding affinity to protein sequence, offering a novel strategy to decode the functional consequences of protein-RNA interactions.

biochemistry↗

bioRxiv2024

Neuropeptides specify and reprogram division of labor in the leafcutter ant Atta cephalotes

Social insects offer powerful models to investigate the mechanistic foundation of elaborate individual behaviors comprising a cooperative community. Workers of the leafcutter ant genus Atta provide an extreme example of behavioral segregation among many phenotypically distinct worker types. We utilize the complex worker system of Atta cephalotes to test the molecular underpinnings of behavioral programming and, in particular, the extent of plasticity to reprogramming. We identify specific neuropeptides as mediators of worker division of labor in A. cephalotes, finding two neuropeptides associated with characteristic behaviors of leafcutting and of brood care. Manipulation via genetic knockdown or by injection of these neuropeptides led to stark loss or gain of each behavior and to transcriptomic shifts in the predicted direction, that is, towards gene pathways expressed in the natural caste. We also compare specific A. cephalotes worker transcriptomes with those of orthologous workers of the eusocial mammal, the naked mole rat H. gaber, revealing global similarities between caste-biased expression and link to specific roles of our studied neuropeptides in ants. This work underscores the essential function of neuropeptides in establishing complex social behavior and a remarkable plasticity among individual behavioral types.

molecular biology↗

bioRxiv2023

A ligation-independent sequencing method reveals tRNA-derived RNAs with blocked 3' termini

Despite the numerous sequencing methods available, the vast diversity in size and chemical modifications of RNA molecules makes the capture of the full spectrum of cellular RNAs a difficult task. By combining quasirandom hexamer priming with a custom template switching strategy, we developed a method to construct sequencing libraries from RNA molecules of any length and with any type of 3 terminal modification, allowing the sequencing and analysis of virtually all RNA species. Ligation-independent detection of all types of RNA (LIDAR) is a simple, effective tool to comprehensively characterize changes in small non-coding RNAs and mRNAs simultaneously, with performance comparable to separate dedicated methods. With LIDAR, we comprehensively characterized the coding and non- coding transcriptome of mouse embryonic stem cells, neural progenitor cells, and sperm. LIDAR detected a much larger variety of tRNA-derived RNAs (tDRs) compared to traditional ligation-dependent sequencing methods, and uncovered the presence of tDRs with blocked 3 ends that had previously escaped detection. Our findings highlight the potential of LIDAR to systematically detect all RNAs in a sample and uncover new RNA species with potential regulatory functions.

genomics↗

bioRxiv2022

Hormonal gatekeeping via the blood brain barrier governs behavior

Here we reveal an unanticipated role of the blood-brain-barrier (BBB) in regulating complex social behavior in ants. Using scRNA-seq we find localization in the BBB of a key hormone-degrading enzyme called Juvenile hormone esterase (Jhe), and we show that this localization governs the level of Juvenile Hormone (JH3) entering the brain. Manipulation of the Jhe level reprograms the brain transcriptome between ant castes. While ant Jhe is retained and functions intracellularly within the BBB, we show that Drosophila Jhe is naturally extracellular. Heterologous expression of ant Jhe into the Drosophila BBB alters behavior in fly to mimic what is seen in ant. Most strikingly, manipulation of Jhe levels in ant reprograms complex behavior between worker castes. Our study thus uncovers a novel, potentially conserved role of the BBB serving as a molecular gatekeeper for a neurohormonal pathway that regulates social behavior.

neuroscience↗

bioRxiv2022

Expanding the epitranscriptomic RNA sequencing and modification mapping mass spectrometry toolbox with field asymmetric waveform ion mobility and electrochemical elution liquid chromatography

Post-transcriptional modifications of RNA strongly influence RNA structure and function. Recent advances in RNA sequencing and mass spectrometry (MS) methods have identified over 140 of these modifications on a wide variety of RNA species. Most next-generation sequencing approaches can only map one RNA modification at a time, and while MS can assign multiple modifications simultaneously in an unbiased manner, MS cannot accurately catalog and assign RNA modifications in complex biological samples due to limitations in fragment length and coverage depth. Thus, a facile method to identify novel RNA modifications while simultaneously locating them in the context of their RNA sequences is still lacking. We combined two orthogonal modes of RNA ion separation before mass-spectrometry identification: high-field asymmetric ion mobility separation (FAIMS) and electrochemically modulated liquid chromatography (EMLC). FAIMS RNA-MS increases both coverage and throughput, while the EMLC LC-MS orthogonally separates RNA of different length and charge. The combination of the two methods offers a broadly applicable platform to improve length and depth of MS-based RNA sequencing while providing contextual access to the analysis of RNA modifications.

biochemistry↗

bioRxiv2022

Transcription factors interact with RNA to regulate genes

Transcription factors (TFs) orchestrate the gene expression programs that define each cells identity. The canonical TF accomplishes this with two domains, one that binds specific DNA sequences and the other that binds protein coactivators or corepressors. We find that at least half of TFs also bind RNA, doing so through a previously unrecognized domain with sequence and functional features analogous to the arginine-rich motif of the HIV transcriptional activator Tat. RNA-binding contributes to TF function by promoting the dynamic association between DNA, RNA and TF on chromatin. TF-RNA interactions are a conserved feature essential for vertebrate development and disrupted in disease. We propose that the ability to bind DNA, RNA and protein is a general property of many TFs and is fundamental to their gene regulatory function.

molecular biology↗

bioRxiv2022

Permethylation of ribonucleosides provides enhanced mass spectrometry quantification of post-transcriptional modifications

Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, degradation, as well as human disease states such as cancer. However, the analysis of ribonucleoside modifications is impeded due to the hydrophilicity of the ribonucleoside molecules. In this research, we used solid-phase permethylation to derivatize the ribonucleosides, and the permethylated ribonucleosides, which were then quantitively analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. The solid-phase permethylation efficiently derivatized the ribonucleosides, and more than 60 RNA modifications were simultaneously monitored using ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. Because of the increased hydrophobicity of permethylated ribonucleosides, this method enhanced retention, separation, and ionization efficiency, resulting in improved detection and quantification when compared to existing analytical strategies of RNA modifications. We applied this new approach to measure the extent of cytosine methylation and hydroxymethylation in RNA obtained from mouse embryonic stem cells with genetic deficiencies in ten-eleven translocation (TET) enzymes. The results matched previously performed analyses and highlighted the sensitivity, efficacy, and robustness of the new method. The advantage of this method enables comprehensive analysis of RNA modifications in biological samples. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/477959v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@a16649org.highwire.dtl.DTLVardef@424b26org.highwire.dtl.DTLVardef@53d701org.highwire.dtl.DTLVardef@48a9cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Intrinsic maturation of sleep output neurons regulates sleep ontogeny in Drosophila

The maturation of sleep behavior across a lifespan (sleep ontogeny) is an evolutionarily conserved phenomenon. Mammalian studies have shown that in addition to increased sleep duration, early life sleep exhibits stark differences compared to mature sleep with regard to the amount of time spent in certain sleep states. How intrinsic maturation of sleep output circuits contributes to sleep ontogeny is poorly understood. The fruit fly Drosophila melanogaster exhibits multifaceted changes to sleep from juvenile to mature adulthood. Here, we use a non-invasive probabilistic approach to investigate changes in sleep architecture in juvenile and mature flies. Increased sleep in juvenile flies is driven primarily by a decreased probability of transitioning to wake, and characterized by more time in deeper sleep states. Functional manipulations of sleep-promoting neurons in the dFB suggest these neurons differentially regulate sleep in juvenile and mature flies. Transcriptomic analysis of dFB neurons at different ages and a subsequent RNAi screen implicate genes involved in distinct molecular processes in sleep control of juvenile and mature flies. These results reveal that dynamic transcriptional states of sleep output neurons contribute to changes in sleep across the lifespan.

neuroscience↗

bioRxiv2021

Genome annotation with long RNA reads reveals new patterns of gene expression in an ant brain

Functional genomic analyses rely on high-quality genome assemblies and annotations. Highly contiguous genome assemblies have become available for a variety of species, but accurate and complete annotation of gene models, inclusive of alternative splice isoforms and transcription start and termination sites remains difficult with traditional approaches. Here, we utilized full-length isoform sequencing (Iso-Seq), a long-read RNA sequencing technology, to obtain a comprehensive annotation of the transcriptome of the ant Harpegnathos saltator. The improved genome annotations include additional splice isoforms and extended 3 untranslated regions for more than 4,000 genes. Reanalysis of RNA-seq experiments using these annotations revealed several genes with caste-specific differential expression and tissue-or caste-specific splicing patterns that were missed in previous analyses. The extended 3 untranslated regions afforded great improvements in the analysis of existing single-cell RNA-seq data, resulting in the recovery of the transcriptomes of 18% more cells. The deeper single-cell transcriptomes obtained with these new annotations allowed us to identify additional markers for several cell types in the ant brain, as well as genes differentially expressed across castes in specific cell types. Our results demonstrate that Iso-Seq is an efficient and effective approach to improve genome annotations and maximize the amount of information that can be obtained from existing and future genomic datasets in Harpegnathos and other organisms.

genomics↗

bioRxiv2019

Social reprogramming in ants induces longevity-associated glia remodeling

Healthy brain aging is of crucial societal importance; however, the mechanisms that regulate it are largely unknown. Social insects are an outstanding model to study the impact of environment and epigenetics on brain function and aging because workers and queens arise from the same genome but display profound differences in behavior and longevity. In Harpegnathos saltator ants, adult workers can transition to a queen-like phenotypic state called gamergate. This caste transition results in reprogramming of social behavior and lifespan extension. Whether these changes in brain function and physiology cause brain remodeling at the cellular level is not known. Using single-cell RNA-sequencing of Harpegnathos brains undergoing caste transition, we uncovered shifts in neuronal and glial populations. In particular, the conversion of workers into long-lived gamergates caused the expansion of ensheathing glia, which maintain brain health by phagocytosing damaged neuronal structures. These glia cells were lost during aging in normal workers but not in longer-lived gamergates. We observed similar caste- and age-associated differences in ensheathing glia in other Hymenoptera as well as Drosophila melanogaster. We propose that enhanced glia-based neuroprotection promotes healthy brain aging and contributes to the extended lifespan of the reproductive caste in social insects.

neuroscience↗

bioRxiv2019

Delineation of the First Human Mendelian Disorder of the DNA Demethylation Machinery: TET3 Deficiency

Germline pathogenic variants in chromatin-modifying enzymes are a common cause of pediatric developmental disorders. These enzymes catalyze reactions that regulate epigenetic inheritance via histone post-translational modifications and DNA methylation. Cytosine methylation of DNA (5mC) is the quintessential epigenetic mark, yet no human Mendelian disorder of DNA demethylation has been delineated. Here, we describe in detail the first Mendelian disorder caused by disruption of DNA demethylation. TET3 is a methylcytosine dioxygenase that initiates DNA demethylation during early zygote formation, embryogenesis, and neuronal differentiation and is intolerant to haploinsufficiency in mice and humans. Here we identify and characterize 11 cases of human TET3 deficiency in 8 families with the common phenotypic features of intellectual disability/global developmental delay, hypotonia, autistic traits, movement disorders, growth abnormalities, and facial dysmorphism. Mono-allelic frameshift and nonsense variants in TET3 occur throughout the coding region. Mono-allelic and bi-allelic missense variants localize to conserved residues with all but one occurring within the catalytic domain and most displaying hypomorphic function in a catalytic activity assay. TET3 deficiency shows substantial phenotypic overlap with other Mendelian disorders of the epigenetic machinery, including intellectual disability and growth abnormalities, underscoring shared disease mechanisms.

genetics↗

bioRxiv2018

Mapping native R-loops genome-wide using a targeted nuclease approach

R-loops are three-stranded DNA:RNA hybrids that are pervasive in the eukaryotic and prokaryotic genomes and have been implicated in a variety of nuclear processes, including transcription, replication, DNA repair, and chromosome segregation. While R-loops may have physiological roles, the formation of stable, aberrant R-loops has been observed in disease, particularly neurological disorders and cancer. Despite the importance of these structures, methods to assess their distribution in the genome invariably rely on affinity purification, which requires large amounts of input material, is plagued by high level of noise, and is poorly suited to capture dynamic and unstable R-loops. Here, we present a new method that leverages the affinity of RNase H for DNA:RNA hybrids to target micrococcal nuclease to genomic sites that contain R-loops, which are subsequently cleaved, released, and sequenced. Our R-loop mapping method, MapR, is as specific as existing techniques, less prone to recover non-specific repetitive sequences, and more sensitive, allowing for genome-wide coverage with low input material and read numbers, in a fraction of the time.

molecular biology↗

bioRxiv2018

Precise re-deposition of nucleosomes on repressive chromatin domains sustain epigenetic inheritance during DNA replication

Chromatin domains and their associated structures must be faithfully inherited through cellular division to maintain cellular identity. Yet, accessing the localized strategies preserving chromatin domain inheritance, specifically the transfer of parental, pre-existing nucleosomes with their associated post-translational modifications (PTMs) during DNA replication is challenging in living cells. We devised an inducible, proximity-dependent labeling system to irreversibly mark replication-dependent H3.1 and H3.2 histone-containing nucleosomes at single desired loci in mouse embryonic stem cells such that their fate after DNA replication could be followed. Strikingly, repressed chromatin domains are preserved through the local re-deposition of parental nucleosomes. In contrast, nucleosomes decorating active chromatin domains do not exhibit such preservation. Notably, altering cell fate leads to an adjustment in the positional inheritance of parental nucleosomes that reflects the corresponding changes in chromatin structure. These findings point to important mechanisms that contribute to parental nucleosome segregation to preserve cellular identity.

biochemistry↗

bioRxiv2017

High-quality genome assemblies uncover caste-specific long non-coding RNAs in ants

Ants are an emerging model system for neuroepigenetics, as embryos with virtually identical genomes develop into different adult castes that display strikingly different physiology, morphology, and behavior. Although a number of ant genomes have been sequenced to date, their draft quality is an obstacle to sophisticated analyses of epigenetic gene regulation. Using long reads generated with Pacific Biosystem single molecule real time sequencing, we have reassembled de novo high-quality genomes for two ant species: Camponotus floridanus and Harpegnathos saltator. The long reads allowed us to span large repetitive regions and join sequences previously found in separate scaffolds, leading to comprehensive and accurate protein-coding annotations that facilitated the identification of a Gp-9-like gene as differentially expressed in Harpegnathos castes. The new assemblies also enabled us to annotate long non-coding RNAs for the first time in ants, revealing several that were specifically expressed during Harpegnathos development and in the brains of different castes. These upgraded genomes, along with the new coding and non-coding annotations, will aid future efforts to identify epigenetic mechanisms of phenotypic and behavioral plasticity in ants.

genomics↗

bioRxiv2017

Olfactory Receptors Are Required For Social Behavior And Neural Plasticity In Ants, As Evidenced By CRISPR-Mediated Gene Knockout

The chemosensory system is key to establishing and maintaining social structure in eusocial insects. Ants exhibit cooperative colonial behaviors reflective of an advanced form of sociality with an extensive dependency on communication. Cuticular hydrocarbons (CHCs) serve as pheromones and cues that regulate multiple aspects of social interactions and behaviors in ants. The perception of CHCs entails odorant receptor neurons (ORNs) that express specific odorant receptors (ORs) encoded by a dramatically expanded Or gene family in ants. Until recently, studies of the biological functions of ORs in eusocial insects were stymied by the lack of genetic tools. In most eusocial insect species, only one or a few queens in a colony can transmit the genetic information to their progeny. In contrast, any worker in the ant Harpegnathos saltator can be converted into a gamergate (pseudo-queen), and used as a foundress to engender an entire new colony and be crossed for genetic experiments. This feature facilitated CRISPR-Cas9 gene targeting to generate a germline mutation in the orco gene that encodes the obligate co-receptor whose mutation should significantly impact ant olfaction. Our results show that Orco exhibits a conserved role in the perception of general odorants but also a role in reproductive physiology and social behavior plasticity in ants. Surprisingly, and in contrast to other insect systems, the loss of OR functionality also dramatically reduces the development of the ant antennal lobe where ORNs project. Taken together, these findings open the possibility of studying the genetics of eusociality and provide inroads towards understanding the function of the expanded ORs family in eusocial insects in regulating caste determination, social communication and neuronal plasticity.

neuroscience↗

bioRxiv2017

A Comparison Between Single Cell RNA Sequencing And Single Molecule RNA FISH For Rare Cell Analysis

The development of single cell RNA sequencing technologies has emerged as a powerful means of profiling the transcriptional behavior of single cells, leveraging the breadth of sequencing measurements to make inferences about cell type. However, there is still little understanding of how well these methods perform at measuring single cell variability for small sets of genes and what \"transcriptome coverage\" (e.g. genes detected per cell) is needed for accurate measurements. Here, we use single molecule RNA FISH measurements of 26 genes in thousands of melanoma cells to provide an independent reference dataset to assess the performance of the DropSeq and Fluidigm single cell RNA sequencing platforms. We quantified the Gini coefficient, a measure of rare-cell expression variability, and find that the correspondence between RNA FISH and single cell RNA sequencing for Gini, unlike for mean, increases markedly with per-cell library complexity up to a threshold of [~]2000 genes detected. A similar complexity threshold also allows for robust assignment of multi-genic cell states such as cell cycle phase. Our results provide guidelines for selecting sequencing depth and complexity thresholds for single cell RNA sequencing. More generally, our results suggest that if the number of genes whose expression levels are required to answer any given biological question is small, then greater transcriptome complexity per cell is likely more important than obtaining very large numbers of cells.

genomics↗