Biology subjects

Schwartz, J. C.

Publications and source records attributed to Schwartz, J. C..

8 recordsLinked to original sources

bioRxiv2025

Bighorn sheep T2T genome assembly reveals differences in immune genes: a potential cause of high morbidity due to respiratory pathogens

The bighorn sheep (Ovis canadensis), despite its close relation to domestic sheep, suffer higher morbidity and mortality from respiratory disease complexes, likely due to genetic differences in immune responses. Unraveling highly repetitive regions such as immune loci and genetic differences was problematic until now. We generated a bighorn sheep telomere-to-telomere assembly, adding 14.28% of novel sequence compared to the previous reference. This enabled the first complete immune loci annotation revealing the IGL and TR loci are significantly short in bighorn sheep. Importantly, a critical immune gene GBP5 and ZNF501, involved in Golgi-mediated immune response, are lacking in bighorn but present in domestic sheep. Re-analysis of a Mycoplasma ovipneumoniae carriage study, using this assembly, identified the immune gene CAPN2 as a key genetic marker for disease carriage, not observable in the original study. This work provides a critical resource for identifying phenotype-linked genetic variation and exploring evolutionary adaptations of bighorn sheep.

genomics↗

bioRxiv2025

Global Profiling of Remodeled Subcellular Structures Due to Drug Treatment and Disease

Cellular biochemistry arises from various interactions between macromolecules, including proteins, nucleic acids, and lipids. These make up membrane-bound organelles, membrane-less compartments, and molecular assemblies and scaffolds. Changes due to stimuli or disease can significantly impact cell fate and metabolism. We recently reported our protocol combining crosslinking and size exclusion chromatography with mass spectrometry (SEC-MS). In this study, we explore global changes to subcellular structure in Ewing sarcoma cell lines or in response to drug treatment. Crosslinking-dependent differential enrichment across four human cell lines is seen for proteins involved in splicing, mitochondria function, and cell division. We confirmed changes to nucleoli structure. We also examine structures affected by a transcription inhibitor, flavopiridol. Following flavopiridol treatment, we observed changes to the levels of transcription and mRNA processing machinery present in large subcellular structures. Unexpected effects were also found, including structural changes to a cytoplasmic organelle, the peroxisome. Along with a reduction in peroxisome function, dissociation of peroxisome pore proteins PEX13 and PEX14 was detected by STORM microscopy. We conclude that SEC-MS combined with crosslinking is a valuable method to detect and quantify drug or disease effects on subcellular structures and may shed light on new aspects to mechanisms underlying their biologic outcomes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/672480v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1f01b55org.highwire.dtl.DTLVardef@9279d4org.highwire.dtl.DTLVardef@7c7492org.highwire.dtl.DTLVardef@c92ff4_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LICrosslinking plus SEC-MS shows global effects on subcellular structure in disease and treatment. C_LIO_LIEwing sarcoma cell lines tested present distinct molecular composition in splicing, mitochondria, and nucleoli. C_LIO_LIA transcription inhibitor flavopiridol disrupts peroxisome function and protein import pores C_LI

biochemistry↗

bioRxiv2024

Ewing Sarcoma Related protein 1 recognizes R-loops by binding DNA forks

EWSR1 (Ewing Sarcoma Related protein 1) is an RNA binding protein that is ubiquitously expressed across cell lines and involved in multiple parts of RNA processing, such as transcription, splicing, and mRNA transport. EWSR1 has also been implicated in cellular mechanisms to control formation of R-loops, a three-stranded nucleic acid structure consisting of a DNA:RNA hybrid and a displaced single-stranded DNA strand. Unscheduled R-loops result in genomic and transcription stress. Loss of function of EWSR1 functions commonly found in Ewing Sarcoma correlates with high abundance of R-loops. In this study, we investigated the mechanism for EWSR1 to recognize an R-loop structure specifically. Using electrophoretic mobility shift assays (EMSA), we detected the high affinity binding of EWSR1 to substrates representing components found in R-loops. EWSR1 specificity could be isolated to the DNA fork region, which transitions between double- and single-stranded DNA. Our data suggests that the Zinc-finger domain (ZnF) with flanking arginine and glycine rich (RGG) domains provide high affinity binding, while the RNA recognition motif (RRM) with its RGG domains offer improved specificity. This model offers a rational for EWSR1 specificity to encompass a wide range in contexts due to the DNA forks always found with R-loops.

biochemistry↗

bioRxiv2023

A highly-contiguous genome assembly of the inbred Babraham pig (Sus scrofa) quantifies breed homozygosity and illuminates porcine immunogenetic variation

The inbred Babraham pig serves as a valuable biomedical model for research due to its high level of homozygosity, including in the major histocompatibility complex (MHC) loci and likely other important immune-related gene complexes, which are generally highly diverse in outbred populations. As the ability to control for this diversity using inbred organisms is of great utility, we sought to improve this resource by generating a long-read whole genome assembly of a Babraham pig. The Babraham genome was de novo assembled using PacBio long-reads and error-corrected using Illumina short-reads. The assembled contigs were then mapped to the current porcine reference assembly, Sscrofa11.1, to generate chromosome-level scaffolds. The resulting Babraham pig assembly is nearly as contiguous as Sscrofa11.1 with a contig N50 of 34.95 Mb and contig L50 of 23. The remaining sequence gaps are generally the result of poor assembly across large and highly repetitive regions such as the centromeres and tandemly duplicated gene families, including immune-related gene complexes, that often vary in gene content between haplotypes. We also further confirm homozygosity across the Babraham pig MHC and characterize the allele content across several immune-related gene complexes, including the contiguous assemblies of the antibody heavy chain locus and leukocyte receptor complex. The Babraham pig genome assembly provides an alternate highly contiguous porcine genome assembly as a resource for the livestock genomics community. The assembly will also aid biomedical and veterinary research that utilizes this animal model such as when controlling for genetic variation is critical.

genomics↗

bioRxiv2022

A role for phase separation to prevent R-loops during transcription

The protein FUS (FUSed in sarcoma) is a metazoan RNA-binding protein that influences RNA production by all three nuclear polymerases. FUS also binds nascent transcripts, RNA processing factors, RNA polymerases, and transcription machinery. We explored the role of FUS binding interactions for activity during transcription. In vitro run-off transcription assays revealed FUS enhanced RNA produced by a non-eukaryote polymerase. Activity also reduced the formation of R-loops between RNA products and their DNA template. Analysis by domain mutation and deletion indicated RNA-binding was required for activity. We interpret that FUS binds and sequesters nascent transcripts to prevent R-loops forming with nearby DNA. DRIP-seq analysis showed that a knockdown of FUS increased R-loop enrichment near expressed genes. Prevention of R-loops by FUS binding to nascent transcripts has potential to affect transcription by any RNA polymerase, highlighting the broad impact FUS can have on RNA metabolism in cells and disease.

biochemistry↗

bioRxiv2020

Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations.

immunology↗

bioRxiv2020

EWS-FLI1 is incorporated into protein granules in cells

Ewing sarcoma is driven by fusion proteins containing a low complexity (LC) domain that is intrinsically disordered and a powerful transcriptional regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing Sarcoma RNA-binding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend Leukemia Virus Integration 1). EWS-FLI1 can bind RNA polymerase II (RNA Pol II) and self-assemble through its low-complexity (LC) domain. The ability of RNA-binding proteins like EWSR1 to self-assemble or phase separate in cells has raised questions about the contribution of this process to EWS-FLI1 activity. We examined EWSR1 and EWS-FLI1 activity in Ewing sarcoma cells by siRNA-mediated knockdown and RNA-seq analysis. More transcripts were affected by the EWSR1 knockdown than expected and these included many EWS-FLI1 regulated genes. We reevaluated physical interactions between EWS-FLI1, EWSR1, and RNA Pol II, and employed a cross-linking based strategy to investigate protein assemblies associated with the proteins. The LC domain of EWS-FLI1 was required for the assemblies observed to form in cells. These results offer new insights into a protein assembly that may enable EWS-FLI1 to bind its wide network of protein partners and contribute to regulation of gene expression in Ewing sarcoma.

biochemistry↗

bioRxiv2020

Determining protein structure by tyrosine bioconjugation

Chemical modification of proteins has been crucial in engineering protein-based therapies, targeted biopharmaceutics, molecular probes, and biomaterials. Here, we explore the use of a conjugation-based approach to sense alternative conformational states in proteins. Tyrosine has both hydrophobic and hydrophilic qualities, thus allowing it to be positioned at protein surfaces, or binding interfaces, or to be buried within a protein. Tyrosine can be conjugated with 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD). We hypothesized that individual protein conformations could be distinguished by labeling tyrosine residues in the protein with PTAD. We conjugated tyrosine residues in a well-folded protein, bovine serum albumin (BSA), and quantified labeled tyrosine with LC-MS/MS. We applied this approach to alternative conformations of BSA produced in the presence of urea. The amount of PTAD labeling was found to relate to the depth of each tyrosine relative to the protein surface. This study demonstrates a new use of tyrosine conjugation using PTAD as an analytic tool able to distinguish the conformational states of a protein.

biochemistry↗