Biology subjects

Ramirez, P.

Publications and source records attributed to Ramirez, P..

10 recordsLinked to original sources

bioRxiv2024

Diverse toxin repertoire but limited metabolic capacities inferred from the draft genome assemblies of three Spiroplasma (Citri clade) strains associated with Drosophila

Spiroplasma (Class Mollicutes) is a diverse wall-less bacterial genus whose members are strictly dependent on eukaryotic hosts (mostly arthropods and plants), with which they engage in pathogenic to mutualistic interactions. Spiroplasma are generally fastidious to culture in vitro, especially those that are vertically transmitted by their hosts, which include flies in the genus Drosophila. Drosophila has been invaded by at least three independent clades of Spiroplasma: Poulsonii (the best studied; contains reproductive manipulators and defensive mutualists associated two major clades of Drosophila; and has among the highest substitution rates within bacteria); Citri (restricted to the repleta group of Drosophila); and Ixodetis. We report the first genome drafts of Drosophila-associated Citri Clade Spiroplasma: strain sMoj from D. mojavensis; strain sAld-Tx from D. aldrichi from Texas (newly discovered; also associated with D. mulleri); and strain sHy2 from D. hydei (the only Drosophila species known to naturally also harbor a Poulsonii clade strain, thereby providing an arena for horizontal gene transfer). Compared to their Poulsonii clade counterparts, we infer that the three Citri clade strains have: (1) equal or worse DNA repair abilities; (b) more limited metabolic capacities, which may underlie their comparatively lower titers and transmission efficiency; and (c) similar content of toxin domains, including at least one ribosome inactivating protein (RIP), which are implicated in the Poulsonii-conferred defense against natural enemies. As a byproduct of our phylogenomic analyses and exhaustive search for certain toxin domains in public databases, we document the toxin repertoire in close relatives of Drosophila-associated Spiroplasma, and in a very divergent newly discovered lineage (i.e., "clade X"). Phylogenies of toxin-encoding genes or domains imply substantial exchanges between closely and distantly related strains. Surprisingly, despite encoding several toxin genes and achieving relatively high prevalences in certain natural populations (sAld-Tx in this study; sMoj in prior work), fitness assays of sMoj (this study) and sAld-Tx (prior work) in the context of wasp parasitism fail to detect a beneficial effect to their hosts. Thus, how Citri clade strains persist in their Drosophila host populations remains elusive. Data summaryAll novel sequencing data are available through National Center for Biotechnology Information (NCBI) repositories. Illumina raw reads, assemblies, and NCBI annotations are available under BioProject Nos. PRJNA506493 for sHy2, PRJNA506491 for sAld-Tx, and PRJNA355307 for sMoj. Oxford Nanopore (MinIon) reads for sHy2 are under SRA Accession Number SRR12348752. Supporting Material is available under the DOI 10.6084/m9.figshare.c.7437997 or as accompanying supporting documents in the corresponding preprint server or scientific journal. Impact statementSymbiotic associations between arthropods and inherited microbes are pervasive, taxonomically and mechanistically diverse, and strongly influential. Research into the mechanisms and processes governing such heritable interactions is hindered by our inability to culture most inherited symbionts outside of their hosts. We studied three heritable strains of Spiroplasma (Citri clade) that naturally associate with Drosophila flies, and that reach relatively high prevalence in certain host populations, but appear to lack traits that would enable them to persist in host populations (e.g. such as high vertical transmission efficiency, reproductive manipulation, or fitness benefits). We compared their genomes to those of a separate Spiroplasma clade (Poulsonii) that associates with Drosophila, which does exhibit some of the traits that contribute to persistence, including protection against natural enemies of their hosts, and also has among the highest DNA substitution rates recorded for bacteria. Compared to Poulsonii, the three Citri clade strains have smaller genomes and fewer genes, leading us to predict they have similarly high DNA substitution rates, but more limited metabolic capacities, which may explain the comparatively lower densities they achieve within individual hosts, and their frequent loss in lab colonies of their hosts. However, the toxin repertoire of Citri clade was comparatively diverse, and the result of horizontal gene exchange among close and distant strains, and within-genome shuffling. We hypothesize that Citri clade strains persist via unknown fitness benefits conferred to their hosts, possibly mediated by toxins, or by substantial horizontal transmission. Our results, which also capitalized on publicly available assemblies, expand the range of Spiroplasma lineages that encode a particular combination of toxin types, and revealed the existence of a highly divergent lineage of Spiroplasma that associates with insects.

genomics↗

bioRxiv2024

Pathogenic tau induces an adaptive elevation in mRNA translation rate at early stages of disease

Alterations in the rate and accuracy of messenger RNA (mRNA) translation are associated with aging and several neurodegenerative disorders, including Alzheimers disease and related tauopathies. We previously reported that error-containing RNA that are normally cleared via nonsense-mediated mRNA decay (NMD), a key RNA surveillance mechanism, are translated in the adult brain of a Drosophila model of tauopathy. In the current study, we find that newly-synthesized peptides and translation machinery accumulate within nuclear envelope invaginations that occur as a consequence of tau pathology, and that the rate of mRNA translation is globally elevated in early stages of disease in adult brains of Drosophila models of tauopathy. Polysome profiling from adult heads of tau transgenic Drosophila reveals the preferential translation of specific mRNA that have been previously linked to neurodegeneration. Unexpectedly, we find that panneuronal elevation of NMD further elevates the global translation rate in tau transgenic Drosophila, as does treatment with rapamycin. As NMD activation and rapamycin both suppress tau-induced neurodegeneration, their shared effect on translation suggests that elevated rates of mRNA translation are an early adaptive mechanism to limit neurodegeneration. Our work provides compelling evidence that tau-induced deficits in NMD reshape the tau translatome by increasing translation of RNA that are normally repressed in healthy cells.

neuroscience↗

bioRxiv2024

Nanopore-based DNA long-read sequencing analysis of the aged human brain

Studies in laboratory models and postmortem human brain tissue from patients with Alzheimers disease have revealed disruption of basic cellular processes such as DNA repair and epigenetic control as drivers of neurodegeneration. While genomic alterations in regions of the genome that are rich in repetitive sequences, often termed "dark regions," are difficult to resolve using traditional sequencing approaches, long-read technologies offer promising new avenues to explore previously inaccessible regions of the genome. In the current study, we leverage nanopore-based long-read whole-genome sequencing of DNA extracted from postmortem human frontal cortex at early and late stages of Alzheimers disease, as well as age-matched controls, to analyze retrotransposon insertion events, non-allelic homologous recombination (NAHR), structural variants and DNA methylation within retrotransposon loci and other repetitive/dark regions of the human genome. Interestingly, we find that retrotransposon insertion events and repetitive element-associated NAHR are particularly enriched within centromeric and pericentromeric regions of DNA in the aged human brain, and that ribosomal DNA (rDNA) is subject to a high degree of NAHR compared to other regions of the genome. We detect a trending increase in potential somatic retrotransposition events of the small interfering nuclear element (SINE) AluY in late-stage Alzheimers disease, and differential changes in methylation within repetitive elements and retrotransposons according to disease stage. Taken together, our analysis provides the first long-read DNA sequencing-based analysis of retrotransposon sequences, NAHR, structural variants, and DNA methylation in the aged brain, and points toward transposable elements, centromeric/pericentromeric regions and rDNA as hotspots for genomic variation.

genomics↗

bioRxiv2024

m6A-dependent circular RNA formation mediatestau-induced neurotoxicity

Circular RNAs (circRNAs), covalently closed RNA molecules that form due to back-splicing of RNA transcripts, have recently been implicated in Alzheimers disease and related tauopathies. circRNAs are regulated by N6-methyladenosine (m6A) RNA methylation, can serve as "sponges" for proteins and RNAs, and can be translated into protein via a cap-independent mechanism. Mechanisms underlying circRNA dysregulation in tauopathies and causal relationships between circRNA and neurodegeneration are currently unknown. In the current study, we aimed to determine whether pathogenic forms of tau drive circRNA dysregulation and whether such dysregulation causally mediates neurodegeneration. We identify circRNAs that are differentially expressed in the brain of a Drosophila model of tauopathy and in induced pluripotent stem cell (iPSC)-derived neurons carrying a tau mutation associated with autosomal dominant tauopathy. We leverage Drosophila to discover that depletion of circular forms of muscleblind (circMbl), a circRNA that is particularly abundant in brains of tau transgenic Drosophila, significantly suppresses tau neurotoxicity, suggesting that tau-induced circMbl elevation is neurotoxic. We detect a general elevation of m6A RNA methylation and circRNA methylation in tau transgenic Drosophila and find that tau-induced m6A methylation is a mechanistic driver of circMbl formation. Interestingly, we find that circRNA and m6A RNA accumulate within nuclear envelope invaginations of tau transgenic Drosophila and in iPSC-derived cerebral organoid models of tauopathy. Taken together, our studies add critical new insight into the mechanisms underlying circRNA dysregulation in tauopathy and identify m6A-modified circRNA as a causal factor contributing to neurodegeneration. These findings add to a growing literature implicating pathogenic forms of tau as drivers of altered RNA metabolism.

neuroscience↗

bioRxiv2021

Preliminary Characterization of Phage-like Particles from the Male-Killing Mollicute Spiroplasma poulsonii (an Endosymbiont of Drosophila)

Bacteriophages are vastly abundant, diverse, and influential, but with few exceptions (e.g. the Proteobacteria genera Wolbachia and Hamiltonella), the role of phages in heritable bacteria-arthropod interactions, which are ubiquitous and diverse, remains largely unexplored. Despite prior studies documenting phage-like particles in the mollicute Spiroplasma associated with Drosophila flies, genomic sequences of such phage are lacking, and their effects on the Spiroplasma-Drosophila interaction have not been comprehensively characterized. We used a density step gradient to isolate phage-like particles from the male-killing bacterium Spiroplasma poulsonii (strains NSRO and MSRO-Br) harbored by Drosophila melanogaster. Isolated particles were subjected to DNA sequencing, assembly, and annotation. Several lines of evidence suggest that we recovered phage-like particles of similar features (shape, size, DNA content) to those previously reported in Drosophila-associated Spiroplasma strains. We recovered three [~]19 kb phage-like contigs (two in NSRO and one in MSRO-Br) containing 21-24 open reading frames, a read-alignment pattern consistent with circular permutation, and terminal redundancy (at least in NSRO). Although our results do not allow us to distinguish whether these phage-like contigs represent infective phage-like particles capable of transmitting their DNA to new hosts, their encoding of several typical phage genes suggests that they are at least remnants of functional phage. We also recovered two smaller non-phage-like contigs encoding a known Spiroplasma toxin (Ribosome Inactivating Protein; RIP), and an insertion element, suggesting that they are packaged into particles. Substantial homology of our particle-derived contigs was found in the genome assemblies of members of the Spiroplasma poulsonii clade.

microbiology↗

bioRxiv2021

Genetic, Genomic and Biophysical Case Study: Familial 46, XY Sex Reversal due to a Novel Inherited Mutation in Human Testis-Determining Factor SRY

ObjectiveTo describe the clinical, histopathological and molecular features of a novel inherited SRY allele (p.Met64Val; consensus box position 9) observed within an extensive pedigree: two 46, XY sisters with primary amenorrhea (16 and 14 years of age; probands P1 and P2), their normal father and brother, and an affected paternal XY grandaunt. Design, Setting, Participants and Outcome MeasurementsFollowing DNA sequencing to identify the SRY mutation, hormonal studies of the probands and histopathological examination of their gonads were performed. Functional consequences of p.Met64Val (and other mutations at this site) were also investigated. ResultsBreast development in P1 and P2 was Tanner II and IV, respectively. Mullerian structures and gonads resembling ovaries were found in each sister. Histopathology revealed gonadal dysgenesis, gonadablastoma and dysgerminoma. AMH/MIS, P450 SCC, and P450 aromatase were expressed in gonadoblastoma tissues. Genomic sequencing revealed no candidate mutations in other genes related to sexual differentiation. Variant p.Met64Val impaired Sox9 transcriptional activation associated with attenuated occupancy of the testis-specific enhancers Enh13 and TESCO. Biophysical studies indicated that the mutant HMG box retains specific DNA binding and DNA bending but with accelerated rate of protein-DNA dissociation. ConclusionThe partial biological activity of p.Met64Val SRY, maintained at the threshold of SRY function, rationalizes opposing paternal and proband phenotypes (the "the father-daughter paradox"). Steroidal biosynthesis by gonadoblastoma may delay genetic diagnosis and recognition of gonadal tumors. Quantitative assessment of inherited SRY alleles highlights the tenuous transcriptional threshold of developmental decision-making in the bipotential gonadal ridge.

developmental biology↗

bioRxiv2021

Pathogenic tau disrupts the cellular program that maintains neuronal identity

Neurons in human Alzheimers disease acquire phenotypes that are also present in various cancers, including over-stabilization of the cytoskeleton, nuclear pleomorphism, decondensation of constitutive heterochromatin, and aberrant activation of the cell cycle. Unlike in cancer, in which cell cycle activation drives tumor formation, activation of the cell cycle in post-mitotic neurons is sufficient to induce neuronal death. Multiple lines of evidence suggest that abortive cell cycle activation is a consequence of pathogenic forms of tau, a protein that drives neurodegeneration in Alzheimers disease and related "tauopathies." We have combined network analysis of human Alzheimers disease and mouse tauopathy with mechanistic studies in Drosophila to discover that pathogenic forms of tau drive abortive cell cycle activation by disrupting the cellular program that maintains neuronal identity. Mechanistically, we identify Moesin, a prognostic biomarker for cancer and mediator of the epithelial-mesenchymal transition (EMT), as a major effector of tau-induced neurotoxicity. We find that aberrant activation of Moesin in neurons acts through the actin cytoskeleton to dysregulate the cellular program that maintains neuronal identity. Our study identifies mechanistic parallels between tauopathy and cancer and sets the stage for novel therapeutic approaches.

neuroscience↗

bioRxiv2021

Pathogenic tau accelerates aging-associated activation of transposable elements in the mouse central nervous system

Transposable elements comprise almost half of the mammalian genome. A growing body of evidence suggests that transposable element dysregulation accompanies brain aging and neurodegenerative disorders, and that transposable element activation is neurotoxic. Recent studies have identified links between pathogenic forms of tau, a protein that accumulates in Alzheimer disease and related tauopathies, and transposable element-induced neurotoxicity. Starting with transcriptomic analyses, we find that age- and tau-induced transposable element activation occurs in the mouse brain. Among transposable elements that are activated at the RNA level in the context of brain aging and tauopathy, we find that the endogenous retrovirus (ERV) class of retrotransposons is particularly enriched. We show that protein encoded by Intracisternal A-particle, a highly active mouse ERV, is elevated in brains of tau transgenic mice. We further demonstrate that brains of tau transgenic mice contain increased DNA copy number of transposable elements, raising the possibility that these elements actively retrotranspose in the context of tauopathy. Taken together, our study lays the groundwork for future mechanistic studies focused on transposable element regulation in the aging mouse brain and in mouse models of tauopathy, while providing support for therapeutic approaches targeting transposable element activation for Alzheimer disease and related tauopathies.

neuroscience↗

bioRxiv2020

Rapid molecular evolution of Spiroplasma symbionts of Drosophila

Spiroplasma are a group of Mollicutes whose members include plant pathogens, insect pathogens, and endosymbionts of animals. Spiroplasma phenotypes have been repeatedly observed to be spontaneously lost in Drosophila cultures, and several studies have documented a high genomic turnover in Spiroplasma symbionts and plant pathogens. These observations suggest that Spiroplasma evolves quickly in comparison to other insect symbionts. Here, we systematically assess evolutionary rates and patterns of Spiroplasma poulsonii, a natural symbiont of Drosophila. We analysed genomic evolution of sHy within flies, and sMel within in vitro culture over several years. We observed that S. poulsonii substitution rates are among the highest reported for any bacteria, and around two orders of magnitude higher compared with other inherited arthropod endosymbionts. The absence of mismatch repair loci mutS and mutL is conserved across Spiroplasma and likely contributes to elevated substitution rates. Further, the closely related strains sMel and sHy (>99.5% sequence identity in shared loci) show extensive structural genomic differences, which potentially indicates a higher degree of host adaptation in sHy, a protective symbiont of Drosophila hydei. Finally, comparison across diverse Spiroplasma lineages confirms previous reports of dynamic evolution of toxins, and identifies loci similar to the male-killing toxin Spaid in several Spiroplasma lineages and other endosymbionts. Overall, our results highlight the peculiar nature of Spiroplasma genome evolution, which may explain unusual features of its evolutionary ecology.

evolutionary biology↗

bioRxiv2019

Effect of heritable symbionts on maternally-derived embryo transcripts

Maternally-transmitted endosymbiotic bacteria are ubiquitous in insects. Among other influential phenotypes, many heritable symbionts of arthropods are notorious for manipulating host reproduction through one of four reproductive syndromes, which are generally exerted during early developmental stages of the host: male feminization; parthenogenesis induction; male killing; and cytoplasmic incompatibility (CI). Major advances have been achieved in understanding mechanisms and identifying symbiont factors involved in reproductive manipulation, particularly male killing and cytoplasmic incompatibility. Nonetheless, whether cytoplasmically-transmitted bacteria influence the maternally-loaded components of the egg or early embryo has not been examined. In the present study, we investigated whether heritable endosymbionts that cause different reproductive phenotypes in Drosophila melanogaster influence the mRNA transcriptome of early embryos. We used mRNA-seq to evaluate differential expression in Drosophila embryos lacking endosymbionts (control) to those harbouring the male-killing Spiroplasma poulsonii strain MSRO-Br, the CI-inducing Wolbachia strain wMel, or Spiroplasma poulsonii strain Hyd1; a strain that lacks a reproductive phenotype and is naturally associated with Drosophila hydei. We found no consistent evidence of influence of symbiont on mRNA composition of early embryos, suggesting that the reproductive manipulation mechanism does not involve alteration of maternally-loaded transcripts. In addition, we capitalized on several available mRNA-seq datasets derived from Spiroplasma-infected Drosophila melanogaster embryos, to search for signals of depurination of rRNA, consistent with the activity of Ribosome Inactivating Proteins (RIPs) encoded by Spiroplasma poulsonii. We found small but statistically significant signals of depurination of Drosophila rRNA in the Spiroplasma treatments (both strains), but not in the symbiont-free control or Wolbachia treatment, consistent with the action of RIPs. The depurination signal was slightly stronger in the treatment with the male-killing strain. This result supports a recent report that RIP-induced damage contributes to male embryo death.

genomics↗