Biology subjects

Yeo, G.

Publications and source records attributed to Yeo, G..

12 recordsLinked to original sources

bioRxiv2025

Unraveling Tyrosine-Kinase Inhibitor Resistance in NSCLC Cells via Same-cell Measurement of RNA, Protein, and Morphological Responses

Non-small cell lung cancer (NSCLC) frequently develops resistance to tyrosine kinase inhibitors (TKIs), limiting the long-term success of targeted therapies. A deeper understanding of resistance mechanisms at the molecular and cellular levels may enable the development of more effective treatment strategies. Here, we applied the Teton detection assay on the AVITI24 platform to measure concurrently RNA, protein, and cellular morphology in NSCLC cell lines treated with the TKIs gefitinib and osimertinib. This single-cell, multiomic analysis revealed distinct expression and morphological profiles between drug-sensitive and resistant cells, including differences in MAPK-related pathway activity. Stratifying responses at the single-cell level uncovered subtle responses not detectable in bulk measurements. We identified CDK4/6 activity as a route of cell survival under TKI treatment and demonstrated that co-treatment with the CDK4/6 inhibitor palbociclib enhanced TKI efficacy. The ability to measure multiomics and cellular morphology in the same cells opens new avenues for future studies aimed at improving personalized treatment strategies in NSCLC and overcoming the obstacles posed by drug resistance.

genomics↗

bioRxiv2024

The YTHDF Proteins Shape the Brain Gene Signatures of Alzheimer's Disease

The gene signatures of Alzheimers Disease (AD) brains reflect an output of a complex interplay of genetic, epigenetic, epi-transcriptomic, and post-transcriptional regulation., yet the dominant factor shaping these signatures remains unclear. To identify the most significant factor that shapes the AD brain signatures, we integrated cellular and molecular features with differential gene expression in in an explainable machine learning framework. Our result indicates that YTHDF proteins, the canonical readers of N6-methyladenosine RNA modification (m6A), are the most influential predictors of the AD brain signatures. We then show that protein modules containing YTHDFs are downregulated in human AD brains, and knocking down and pharmacologically inhibiting YTHDFs in iPSC-derived 2D and 3D neuronal models recapitulate the AD-associated transcriptional signatures. Furthermore, eCLIP-seq analysis revealed that YTHDF proteins influence AD signatures through both m6A-dependent and independent pathways. These results highlight the central role of YTHDF proteins in shaping the gene signatures of AD brains.

neuroscience↗

bioRxiv2023

Fetally-encoded GDF15 and maternal GDF15 sensitivity are major determinants of nausea and vomiting in human pregnancy

Human pregnancy is frequently accompanied by nausea and vomiting that may become severe and life-threatening, as in hyperemesis gravidarum (HG), the cause of which is unknown. Growth Differentiation Factor-15 (GDF15), a hormone known to act on the hindbrain to cause emesis, is highly expressed in the placenta and its levels in maternal blood rise rapidly in pregnancy. Variants in the maternal GDF15 gene are associated with HG. Here we report that fetal production of GDF15, and maternal sensitivity to it, both contribute substantially to the risk of HG. We found that the great majority of GDF15 in maternal circulation is derived from the feto-placental unit and that higher GDF15 levels in maternal blood are associated with vomiting and are further elevated in patients with HG. Conversely, we found that lower levels of GDF15 in the non-pregnant state predispose women to HG. A rare C211G variant in GDF15 which strongly predisposes mothers to HG, particularly when the fetus is wild-type, was found to markedly impair cellular secretion of GDF15 and associate with low circulating levels of GDF15 in the non-pregnant state. Consistent with this, two common GDF15 haplotypes which predispose to HG were associated with lower circulating levels outside pregnancy. The administration of a long-acting form of GDF15 to wild-type mice markedly reduced subsequent responses to an acute dose, establishing that desensitisation is a feature of this system. GDF15 levels are known to be highly and chronically elevated in patients with beta thalassemia. In women with this disorder, reports of symptoms of nausea or vomiting in pregnancy were strikingly diminished. Our findings support a causal role for fetal derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by pre-pregnancy exposure to GDF15, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.

genetics↗

bioRxiv2023

Systematic elucidation of genetic mechanisms underlying cholesterol uptake

Genetic variation contributes greatly to LDL cholesterol (LDL-C) levels and coronary artery disease risk. By combining analysis of rare coding variants from the UK Biobank and genome-scale CRISPR-Cas9 knockout and activation screening, we have substantially improved the identification of genes whose disruption alters serum LDL-C levels. We identify 21 genes in which rare coding variants significantly alter LDL-C levels at least partially through altered LDL-C uptake. We use co-essentiality-based gene module analysis to show that dysfunction of the RAB10 vesicle transport pathway leads to hypercholesterolemia in humans and mice by impairing surface LDL receptor levels. Further, we demonstrate that loss of function of OTX2 leads to robust reduction in serum LDL-C levels in mice and humans by increasing cellular LDL-C uptake. Altogether, we present an integrated approach that improves our understanding of genetic regulators of LDL-C levels and provides a roadmap for further efforts to dissect complex human disease genetics.

genetics↗

bioRxiv2022

Proteomic discovery of chemical probes that perturb protein complexes in human cells

Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.

biochemistry↗

bioRxiv2020

Elucidation of remdesivir cytotoxicity pathways through genome-wide CRISPR-Cas9 screening and transcriptomics

The adenosine analogue remdesivir has emerged as a frontline antiviral treatment for SARS-CoV-2, with preliminary evidence that it reduces the duration and severity of illness1. Prior clinical studies have identified adverse events1,2, and remdesivir has been shown to inhibit mitochondrial RNA polymerase in biochemical experiments7, yet little is known about the specific genetic pathways involved in cellular remdesivir metabolism and cytotoxicity. Through genome-wide CRISPR-Cas9 screening and RNA sequencing, we show that remdesivir treatment leads to a repression of mitochondrial respiratory activity, and we identify five genes whose loss significantly reduces remdesivir cytotoxicity. In particular, we show that loss of the mitochondrial nucleoside transporter SLC29A3 mitigates remdesivir toxicity without a commensurate decrease in SARS-CoV-2 antiviral potency and that the mitochondrial adenylate kinase AK2 is a remdesivir kinase required for remdesivir efficacy and toxicity. This work elucidates the cellular mechanisms of remdesivir metabolism and provides a candidate gene target to reduce remdesivir cytotoxicity.

genetics↗

bioRxiv2020

Selecting Student-Authored Questions for Summative Assessments

Production of high-quality multiple-choice questions (MCQs) for both formative and summative assessment is a time-consuming task requiring great skill, creativity, and insight. The transition to online examinations, with the concomitant exposure of previously tried-and-tested MCQs, exacerbates the challenges of question production, and highlights the need for innovative solutions. Several groups have shown that it is practical to leverage the student cohort to produce a very large number of syllabus-aligned MCQs for study banks. Although student-generated questions are well suited for formative feedback and practice activities, they are generally not thought to be suitable for high-stakes assessments. In this study, we aimed to demonstrate that training can be provided to students in a scalable fashion to generate questions of similar quality to those produced by experts, and that identification of suitable questions can be achieved with minimal academic review and editing. Biochemistry and Molecular Biology students were assigned a series of activities designed to coach them in the art of writing and critiquing MCQs. This training resulted in the production of over one thousand MCQs that were then gauged for potential by either expert academic judgement, or via a data-driven approach in which the questions were trialled objectively in a low-stakes test. Questions selected by either method were then deployed in a high-stakes in-semester assessment alongside questions from two academically authored sources: textbook-derived MCQs, and past paper questions. A total of 120 MCQs from these four sources were deployed in assessments attempted by over 600 students. Each question was subjected to rigorous performance analysis, including the calculation of standard metrics from classical test theory and more sophisticated Item Response Theory (IRT) measures. The results showed that MCQs authored by students and selected at low cost performed as well as questions authored by academics, illustrating the potential of this strategy for the efficient creation of large numbers of high quality MCQs for summative assessment.

scientific communication and education↗

bioRxiv2020

Transcriptome-wide profiles of circular RNA and RNA binding protein interactions reveal effects on circular RNA biogenesis and cancer pathway expression

Circular RNAs (circRNAs) are stable, often highly expressed RNA transcripts with potential to modulate other regulatory RNAs. A few circRNAs have been shown to bind RNA binding proteins (RBPs), however, little is known about the prevalence and strength of these interactions in different biological contexts. Here, we comprehensively evaluate the interplay between circRNAs and RBPs in the ENCODE cell lines, HepG2 and K562, by profiling the expression of circRNAs in fractionated total RNA-sequencing samples and analyzing binding sites of 150 RBPs in large eCLIP data sets. We show that KHSRP binding sites are enriched in flanking introns of circRNAs in both HepG2 and K562 cells, and that KHSRP depletion affects circRNA biogenesis. Additionally, we show that exons forming circRNAs are generally enriched with RBP binding sites compared to non-circularizing exons. To detect individual circRNAs with regulatory potency, we computationally identify circRNAs that are highly covered by RBP binding sites and experimentally validate circRNA-RBP interactions by RNA immunoprecipitations. We characterize circCDYL, a highly expressed circRNA with clinical and functional implications in bladder cancer, which is covered with GRWD1 binding sites. We confirm that circCDYL binds GRWD1 in vivo and functionally characterizes the effect of circCDYL-GRWD1 interactions on target genes in HepG2. Furthermore, we confirm interactions between circCDYL and RBPs in bladder cancer cells and demonstrate that circCDYL depletion affects hallmarks of cancer and perturbs the expression of key cancer genes, e.g. TP53 and MYC. Finally, we show that elevated levels of highly RBP-covered circRNAs, including circCDYL, are associated with overall survival of bladder cancer patients. Our study demonstrates transcriptome-wide and cell-type-specific circRNA-RBP interactions that could play important regulatory roles in tumorigenesis.

genomics↗

bioRxiv2020

Molecular determinants and heterogeneity of tissue-resident memory CD8+ T lymphocytes revealed by single-cell RNA sequencing

During an immune response to microbial infection, CD8+ T cells give rise to distinct classes of cellular progeny that coordinately mediate clearance of the pathogen and provide long-lasting protection against reinfection, including a subset of non-circulating tissue-resident memory (TRM) cells that mediate potent protection within non-lymphoid tissues. Here, we utilized single-cell RNA-sequencing to examine the gene expression patterns of individual CD8+ T cells in the spleen and small intestine intraepithelial lymphocyte (siIEL) compartment throughout the course of their differentiation in response to viral infection. These analyses revealed previously unknown transcriptional heterogeneity within the siIEL CD8+ T cell population at several states of differentiation, representing functionally distinct TRM cell subsets as well as a subset of TRM cell precursors within the tissue early in infection. Taken together, these findings may inform strategies to optimize CD8+ T cell responses to protect against microbial infection and cancer. One sentence summaryHere, we applied single-cell RNA-sequencing to elucidate the gene expression patterns of individual CD8+ T cells differentiating throughout the course of infection in the spleen and small intestinal epithelium, which revealed previously unidentified molecular determinants of tissue-resident T cell differentiation as well as functional heterogeneity within the tissue-resident CD8+ T cell population.

immunology↗

bioRxiv2018

Modulation of RNA-dependent interactions in stress granules prevents persistent TDP-43 accumulation in ALS/FTD

Human genetic variants are usually represented by four values with variable length: chromosome, position, reference and alternate alleles. Thereis no guarantee that these components are represented in a consistent way across different data sources, and processing variant-based data can be inefficient because four different comparison operations are needed for each variant, three of which are string comparisons. Working with strings, in contrast to numbers, poses extra challenges on computer memory allocation and data-representation. Existing variant identifiers do not typicallyrepresent every possible variant we may be interested in, nor they are directly reversible. To overcome these limitations, VariantKey, a novel reversible numerical encoding schema for human genetic variants, is presented here alongside a multi-language open-source software implementation (http://github.com/genomicspls/variantkey). VariantKey represents variants as single 64 bit numeric entities, while preserving the ability to be searched and sorted by chromosome and position. The individual components of short variants can be directly read back from the VariantKey, while long variants are supported with a fast lookup table.\n\nHighlightsO_LI~100 compounds identified by high-content screen inhibit SGs in HEK293, NPCs and iPS-MNs.\nC_LIO_LIALS-associated RBPs are recruited to SGs in an RNA-dependent manner\nC_LIO_LIMolecules with planar moieties prevent recruitment of ALS-associated RBPs to SGs\nC_LIO_LICompounds inhibit TDP-43 accumulation in SGs and in TARDBP mutant iPS-MNs.\nC_LI

neuroscience↗

bioRxiv2018

Active protein neddylation or ubiquitylation is dispensable for stress granule dynamics

Many protein homeostasis stressors induce the formation of membraneless cytoplasmic stress granules (SGs) that contain large assemblies of repressed mRNAs and associated RNA binding proteins. Similar stressors have been shown to globally alter the function of the ubiquitin proteasome system (UPS) resulting in the accumulation of ubiquitylated proteins. Previous studies have demonstrated that ubiquitin and specific UPS components co-localize with SGs and that reducing the abundance or activity of ubiquitin pathway proteins can inhibit SG formation. These studies suggest that SG dynamics and composition may be regulated by ubiquitylation of SG resident proteins. Using ubiquitin-specific proteomic approaches, we demonstrate that many proteins, including some SG proteins are dynamically ubiquitylated upon SG-inducing sodium arsenite treatment. We utilized potent and selective inhibitors of the ubiquitin activating enzyme (UAE) or the NEDD8 activating enzyme (NAE) to directly test if active protein ubiquitylation or neddylation was required for SG dynamics. Using ubiquitin-site specific proteomics, we establish that UAE inhibition results in the rapid loss of nearly all protein ubiquitylation regardless of ubiquitin chain type. Addition of UAE or NAE inhibitors to cells did not alter arsenite-induced SG formation or dissolution. While we confirmed that ubiquitin co-localizes with both sodium arsenite and thapsigargin-induced SGs, antibodies that recognize all forms of ubiquitin more strongly co-localize with SGs compared to antibodies that preferentially recognize polyubiquitin or specific polyubiquitin-linkages. Interestingly, ubiquitin itself co-localizes with SGs in a UAE independent manner suggesting that the ubiquitin present within SGs is likely unconjugated ubiquitin. Our findings clearly demonstrate that active protein ubiquitylation or neddylation is not required for SG dynamics. These results suggest that ubiquitin-binding SG proteins may recruit free ubiquitin into SGs to modulate SG protein interactions.

cell biology↗

bioRxiv2017

Proximity labeling reveals an extensive steady-state stress granule interactome and insights to neurodegeneration

Stress granules (SGs) are transient ribonucleoprotein (RNP) aggregates that form in response to proteotoxic stress. Although SGs are distinct from aggregates observed in neurodegenerative disorders, they share protein components. We used APEX-mediated proximity labeling combined with quantitative mass spectrometry and high-throughput imaging to identify >100 previously unknown SG proteins in human cells, about 10% of which localize to SGs in a cell type- or stress type-dependent manner. Supporting a link between SG proteins and neurodegeneration, we demonstrate aberrant SG composition and subcellular distribution in iPSC-derived motor neurons from ALS patients, and identify several known and previously unidentified SG proteins that modify toxicity of mutant FUS and TDP-43 overexpression in Drosophila. We show that even in an unstressed steady-state, SG proteins form a densely-connected protein interaction network (PIN) and propose a model in which existing RNPs coalesce rapidly into microscopically visible granules that can act as gateways to pathological protein aggregation.\n\nHighlights O_LIAPEX proximity labeling of dynamic RNP granules identifies over 100 novel SG proteins\nC_LIO_LISG proteins form a densely-connected protein interaction network in unstressed cells\nC_LIO_LISystematic immunofluorescence analysis reveals stress- and cell type-specific SG composition\nC_LIO_LIALS motor neurons contain SGs with distinct content and subcellular distribution\nC_LI

cell biology↗