Biology subjects

Ebmeier, C. C.

Publications and source records attributed to Ebmeier, C. C..

16 recordsLinked to original sources

bioRxiv2026

Structure-guided antisense-oligonucleotides selectively modulate frameshifting of a human gene

Programmed -1 ribosomal frameshifting (-1 PRF) is a conserved translational recoding mechanism that expands proteomic diversity and regulates gene expression through RNA structural elements, most notably stimulatory pseudoknots. This mechanism is common in viruses, where it is used to control stoichiometry of viral protein products generated by the host cell to direct viral replication. Despite its biological importance, strategies to selectively modulate frameshifting remain limited. The mammalian retrotransposon-derived gene PEG10 also relies on -1 PRF to produce a fusion protein, gag-pol, which is necessary for reproduction but has also been implicated in neurological diseases. Here, we establish an antisense oligonucleotide (ASO) targeting an RNA structural element as an effective approach to tune PEG10 frameshifting. Using structure prediction, systematic antisense tiling across the PEG10 pseudoknot, and multiple model systems, we identify a discrete functional hotspot within the lower RNA stem that governs frameshift efficiency. ASOs targeting this region selectively suppress gag-pol production with minimal impact on gag, thereby shifting the ratio of protein products in a dose-dependent manner. Mechanistic dissection using RNase H-active and -inactive ASO designs, pre-annealed duplexes, and fluorescence-based subcellular localization supports a predominantly nuclear mode of action in which ASOs engage nascent PEG10 transcripts and bias pseudoknot folding away from the frameshift-competent conformation. Functional effects are conserved between human cell lines and murine models, including neurons, highlighting the generality of this strategy. Together, our results define RNA structural dynamics as a druggable layer of translational regulation and establish antisense modulation of pseudoknot folding as a way to control endogenous frameshifting. This work provides a conceptual and practical framework for targeting recoding-dependent gene products such as PEG10 in disease and suggests broader applicability of structure-directed ASOs to viral and cellular frameshifting elements.

biochemistry↗

bioRxiv2025

Identification of proteins exhibiting in vitro RNA chaperone activity through gradient profiling in the Lyme disease spirochete, Borrelia burgdorferi

RNA-binding proteins (RBPs) play key roles in regulating gene expression in bacteria. However, relatively few RBPs have been discovered and characterized in Borrelia (Borreliella) burgdorferi, the causative agent of Lyme disease. We utilized gradient profiling to identify putative RBPs that co-sediment with small RNAs (sRNAs) and nascent mRNAs. We employed in vitro and in vivo assays to characterize the RNA chaperone activities of several proteins we identified in the gradient profiling. The previously hypothetical proteins BB0749, BB0713 and BB0796, as well as the chemotaxis-related protein CheY2 and the flagella-associated protein FlgV displayed RNA annealing and/or strand displacement activity. Moreover, in vivo Co-IP assays demonstrated BB0749 binds RNA in B. burgdorferi.

molecular biology↗

bioRxiv2025

Cytoplasmic CaMKIIδ-B prevents myocardial recovery in heart failure

Restoration of cardiac function in patients with advanced heart failure is rare, and the molecular processes that regulate recovery are unknown. To identify potential mechanisms, we studied paired myocardial samples before and after left ventricular assist device therapy, where significant cardiac functional recovery occurred in [~]25% of patients. We found that expression of the nuclear B isoform of Ca2+/calmodulin-dependent protein kinase II{delta} (CaMKII{delta}-B) inversely correlated with recovery. Furthermore, increased phosphorylation near the CaMKII{delta}-B nuclear localization signal in non-responders prevented its auto-activation dependent nuclear translocation. Expression of a cytoplasm-restricted CaMKII{delta}-B in cardiomyocytes dramatically remodeled the phospho-proteome and impaired contractility, while a nuclear-competent version did not. Modulating CaMKII{delta} subcellular localization may thus represent a therapeutic strategy for advanced heart failure.

physiology↗

bioRxiv2025

Dynamic hyperplastic cardiac growth in Burmese pythons

Cardiomyocytes hyperplasia is the primary form of fetal heart growth, whereas this proliferative capacity is largely lost in adults across most species. The limited ability of adult cardiomyocytes to re-enter the cell cycle is a major cause of cardiac injury-induced morbidity and mortality. Here, we report that post-prandial Burmese python cardiomyocytes activate cell cycle re-entry to promote persistent cardiac growth. Burmese pythons normally eat large meals infrequently, resulting in reversible cardiac hypertrophy. We found that frequent feeding of large meals amplifies the modest post-prandial cardiac proliferation identified in an infrequent feeding interval. By activating E2F and Forkhead Box M1 (FoxM1) pro-proliferation transcriptional networks, frequently fed Burmese pythons initiate cardiomyocyte hyperplasia. These findings identify hyperplasia as a natural means of sustained cardiac growth in Burmese pythons and support the use of pythons as a new model for investigating proliferative cardiac remodeling.

cell biology↗

bioRxiv2025

Multi-omics and biochemical reconstitution reveal CDK7-dependent mechanisms controlling RNA polymerase II function at gene 5'- and 3'-ends

CDK7 regulates RNA polymerase II (RNAPII) initiation, elongation, and termination through incompletely understood mechanisms. Because contaminating kinases precluded CDK7 analysis with nuclear extracts, we completed biochemical assays with purified factors. Reconstitution of RNAPII transcription initiation showed CDK7 inhibition slowed and/or paused RNAPII promoter-proximal transcription, which reduced re-initiation. These CDK7-regulatory functions were Mediator- and TFIID-dependent. Similarly in human cells, CDK7 inhibition reduced transcription by suppressing RNAPII activity at promoters, consistent with reduced initiation and/or re-initiation. Moreover, widespread 3-end readthrough transcription was observed in CDK7-inhibited cells; mechanistically, this occurred through rapid nuclear depletion of RNAPII elongation and termination factors, including high-confidence CDK7 targets. Collectively, these results define how CDK7 governs RNAPII function at gene 5-ends and 3-ends, and reveal that nuclear abundance of elongation and termination factors is kinase-dependent. Because 3-readthrough transcription is commonly induced during stress, our results further suggest regulated suppression of CDK7 activity may enable this RNAPII transcriptional response.

biochemistry↗

bioRxiv2024

Cyanoexosortase B is essential for motility, biofilm formation and scytonemin production in a filamentous cyanobacterium

Exosortases are involved in trafficking proteins containing PEP-CTERM domains to the exterior of gram-negative bacterial cells. The role of these proteins in cyanobacteria, where such homologs are common, has not been defined. The filamentous cyanobacterium Nostoc punctiforme contains a single putative exosortase, designated cyanoexosortase B (CrtB), implicated by previous work in both the motility, and in the production of the UV-absorbing pigment, scytonemin. To determine the role of crtB in N. punctiforme, a crtB-deletion strain ({Delta}crtB) was generated. {Delta}crtB presented the loss of motility, biofilm formation, and scytonemin production. In the case of motility, the {Delta}crtB mutant exhibited a specific defect in the ability of hormogonia (specialized motile filaments) to adhere to hormogonium polysaccharide (HPS) and several PEP-CTERM proteins expressed in motile hormogonia were differentially abundant in the exoproteome of the wild type compared to the {Delta}crtB strain. These results are consistent with the hypothetical role of CrtB in the processing and export of PEP-CTERM proteins that play a critical role in stabilizing the interaction between the filament surface and HPS to facilitate motility and biofilm formation. In the case of scytonemin, the late biosynthetic steps of which occur in the periplasm and whose operon contains several putative PEP-CTERM proteins, {Delta}crtB failed to produce it. Given the abundance of putative PEP-CTERM proteins encoded in the N. punctiforme genome, and the fact that this study only associates a fraction of them with biological functions, it seems likely that CrtB may play an important role in other biological processes in cyanobacteria. ImportanceIn gram-negative bacteria, exosortases facilitate the trafficking of proteins to the exterior of the cell where they have been implicated in stabilizing the association of extracellular polymeric substances (EPS) with the cell surface to facilitate biofilm formation and flocculation, but the role of exosortases in cyanobacteria has not been explored. Here, we characterize the role of cyanoexosortase B (CrtB) in the filamentous cyanobacterium Nostoc punctiforme, demonstrating that crtB is essential for motility, biofilm formation, and the production of the sunscreen pigment scytonemin. These findings have important implications for understanding motility and biofilm formation in filamentous cyanobacteria as well as efforts toward heterologous production of scytonemin in non-native hosts.

microbiology↗

bioRxiv2024

Digital Light Processing 3D Printing enables High Throughput Fabrication of Human Engineered Heart Tissues for Disease Modeling

3D in vitro engineered heart tissue (EHT) models recapitulate aspects of native cardiac physiology but are often limited by scalability, cost, and reproducibility. Here, we report a simple, one-step method for rapid ([~]minutes) fabrication of molds using digital light processing (DLP)-based 3D printing that support the formation of EHTs by human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with high reproducibility (>95% efficiency) and varied designs (e.g., length, aspect ratio). Compared to 2D iPSC-CMs, 3D EHTs display enhanced maturity, including increased expression of {beta}-oxidation genes, higher concentrations of sarcomeric myosins, improved sarcomere density and alignment, and enrichment of cardiac pathways (e.g., upregulation of sodium channels, action potentials, contraction). The technology is applied to model pathological cardiac hypertrophy in vitro, using either (i) acute adrenergic agonism or (ii) chronic culture within stiff hydrogel molds. Treated EHTs exhibit increased levels of pathology-associated gene expression and activation of signaling cascades involved in pathological remodeling compared to untreated controls or treated 2D iPSC-CMs. Thus, our method results in robust yet simpler, cheaper, and faster EHTs to study cardiac disease.

bioengineering↗

bioRxiv2024

An EYA3/NF-κB/CCL2 signaling axis suppresses cytotoxic NK cells in the pre-metastatic niche to promote triple negative breast cancer metastasis

Patients with Triple Negative Breast Cancer (TNBC) exhibit high rates of metastases and poor prognoses. The Eyes absent (EYA) family of proteins are developmental transcriptional cofactors/phosphatases that are re-expressed and/or upregulated in numerous cancers. Herein, we demonstrate that EYA3 correlates with decreased survival in breast cancer, and that it strongly, and specifically, regulates metastasis via a novel mechanism that involves NF-kB signaling and an altered innate immune profile at the pre-metastatic niche (PMN). Remarkably, restoration of NF-kB signaling downstream of Eya3 knockdown (KD) restores metastasis without restoring primary tumor growth, isolating EYA3/NF-kB effects to the metastatic site. We show that secreted CCL2, regulated downstream of EYA3/NF-kB, specifically decreases cytotoxic NK cells in the PMN and that re-expression of Ccl2 in Eya3-KD cells is sufficient to rescue activation/levels of cytotoxic NK cells in vitro and at the PMN, where EYA3-mediated decreases in cytotoxic NK cells are required for metastatic outgrowth. Importantly, analysis of public breast cancer datasets uncovers a significant correlation of EYA3 with NF-kB/CCL2, underscoring the relevance of EYA3/NF-kB/CCL2 to human disease. Our findings suggest that inhibition of EYA3 could be a powerful means to re-activate the innate immune response at the PMN, inhibiting TNBC metastasis. SignificanceEYA3 promotes metastasis of TNBC cells by promoting NF-kB-mediated CCL2 expression and inhibiting cytotoxic NK cells at the pre-metastatic niche, highlighting a potential therapeutic target in this subset of breast cancer.

cancer biology↗

bioRxiv2024

A Conserved Mechanism of Cardiac Hypertrophy Regression through FoxO1

The heart is a highly plastic organ that responds to diverse stimuli to modify form and function. The molecular mechanisms of adaptive physiological cardiac hypertrophy are well-established; however, the regulation of hypertrophy regression is poorly understood. To identify molecular features of regression, we studied Burmese pythons which experience reversible cardiac hypertrophy following large, infrequent meals. Using multi-omics screens followed by targeted analyses, we found forkhead box protein O1 (FoxO1) transcription factor signaling, and downstream autophagy activity, were downregulated during hypertrophy, but re-activated with regression. To determine whether these events were mechanistically related to regression, we established an in vitro platform of cardiomyocyte hypertrophy and regression from treatment with fed python plasma. FoxO1 inhibition prevented regression in this system, while FoxO1 activation reversed fed python plasma-induced hypertrophy in an autophagy-dependent manner. We next examined whether FoxO1 was implicated in mammalian models of reversible hypertrophy from exercise and pregnancy and found that in both cases FoxO1 was activated during regression. In these models, as in pythons, activation of FoxO1 was associated with increased expression FoxO1 target genes involved in autophagy. Taken together, our findings suggest FoxO1-dependent autophagy is a conserved mechanism for regression of physiological cardiac hypertrophy across species. HIGHLIGHTSO_LIPost-prandial cardiac remodeling in Burmese pythons is associated with dynamic regulation of FoxO1 and autophagy. C_LIO_LIRegression of fed python plasma-induced hypertrophy in mammalian cardiomyocytes is FoxO1-dependent. C_LIO_LIActivation of FoxO1-dependent autophagy is a conserved feature of physiological hypertrophy regression in mammalian models. C_LI

physiology↗

bioRxiv2023

The transcriptomic and proteomic ramifications of segmental amplification

Gene amplification can drive adaptation by rapidly increasing the cellular dosage of critical gene products. Segmental amplifications often encompass large genomic regions surrounding the gene(s) under selection for higher dosage. Overexpression of co-amplified neighboring genes imposes a substantial metabolic burden. While compensatory mutations can decrease inappropriate overexpression of co-amplified genes, it takes time for such mutations to arise. The extent to which intrinsic regulatory mechanisms modulate expression of co-amplified genes in the immediate aftermath of segmental amplification is largely unknown. To address the collateral effects of segmental amplification, we evolved replicate cultures of an Escherichia coli mutant under conditions that select for higher dosage of an inefficient enzyme whose weak activity limits growth rate. Segmental amplifications encompassing the gene encoding the weak-link enzyme arose in all populations. Amplicons ranged in size (9 to 125 kb) and copy number (2 to 12 copies). We performed RNA-seq and label-free proteomics to quantify expression of amplified genes present at 2, 6, and 12 copies. mRNA expression generally scales with gene copy number, but protein expression scales less well with both gene copy number and mRNA expression. We characterize the molecular mechanisms underlying discrepancies between gene copy number and expression for several cases. We also show that segmental amplifications can have system-wide consequences by indirectly altering expression of non-amplified genes. Our findings indicate that the fitness benefit derived from segmental amplification depends on the combined effects of amplification size, gene content, and copy number as well as collateral effects on non-amplified genes. Significance StatementGene amplification frequently drives rapid adaptation when organisms are challenged by harsh conditions. However, gene amplification rarely amplifies only the gene(s) under selection for higher dosage. Segmental amplifications often include many co-amplified neighbor genes. Because segmental amplifications can reach very high copy number, overexpression of co-amplified genes can impose an energetic burden and perturb physiology, yet this aspect of gene amplification has received little attention. Here, we performed transcriptomic and proteomic analysis of laboratory-evolved Escherichia coli strains containing large, high-copy-number amplifications. We found that mRNA levels, but often not protein levels, scale well with gene copy number. We identified amplified genes that exhibit discrepancies between gene copy number and mRNA/protein expression and examine the molecular mechanisms underlying these discrepancies.

evolutionary biology↗

bioRxiv2022

UBQLN2 restrains the domesticated retrotransposon PEG10 to maintain neuronal health in ALS

Amyotrophic Lateral Sclerosis (ALS) is a fatal, neurodegenerative disease characterized by progressive motor neuron dysfunction and loss. A portion of ALS is caused by mutation of the proteasome shuttle factor Ubiquilin 2 (UBQLN2), but the molecular pathway leading from UBQLN2 dysfunction to disease remains unclear. Here, we demonstrate a function of UBQLN2 in regulating activity of the domesticated gag-pol retrotransposon paternally expressed gene 10 (PEG10) in human cells and tissues. In cells, the PEG10 gag-pol protein cleaves itself in a mechanism reminiscent of retrotransposon self-processing to generate a liberated nucleocapsid fragment, which uniquely localizes to the nucleus and changes expression of genes involved in axon remodeling. In spinal cord tissue from ALS patients, PEG10 gag-pol is elevated compared to healthy controls. These findings implicate the retrotransposon-like activity of PEG10 as a contributing mechanism in ALS through regulation of gene expression, and restraint of PEG10 as a primary function of UBQLN2.

cell biology↗

bioRxiv2020

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs

Motile cilia and flagella are built from stable populations of doublet microtubules that comprise their axonemes. Their unique stability is brought about, at least in part, by a network of Microtubule Inner Proteins (MIPs) found in the lumen of their doublet microtubules. Rib72A and Rib72B were identified as microtubule inner proteins (MIPs) in the motile cilia of Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of multiple MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout (KO) Tetrahymena cells. From this analysis we identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

molecular biology↗

bioRxiv2020

Multi-Omic Analysis Reveals Cannabidiol Disruption of Cholesterol Homeostasis in Human Cell Lines

The non-psychoactive cannabinoid, cannabidiol (CBD), is FDA-approved for treatment of two drug-resistant epileptic disorders, and is seeing increased use among the general public, yet the mechanisms that underlie its therapeutic effects and side-effect profiles remain unclear. Here, we report a systems-level analysis of CBD action in human cell lines using temporal multi-omic profiling. FRET-based biosensor screening revealed that CBD treatment resulted in a sharp rise in cytosolic calcium, and activation of AMPK and ERK kinases in human keratinocyte and neuroblastoma cell lines. CBD treatment led to alterations in the abundance of metabolites, mRNA transcripts, and proteins consistent with activation of cholesterol biosynthesis, transport and storage. We found that CBD rapidly incorporated into cellular membranes and altered cholesterol chemical activity, suggesting direct perturbation of cholesterol-dependent membrane properties. CBD treatment induced apoptosis in a dose-dependent manner in multiple human cell lines, which was rescued by inhibition of cholesterol synthesis, and potentiated by compounds that disrupt cholesterol trafficking and storage. Our data point to a pharmacological interaction of CBD with cholesterol homeostasis pathways, with potential implications in its therapeutic use.

systems biology↗

bioRxiv2019

Mutations that improve the efficiency of a weak-link enzyme are rare compared to adaptive mutations elsewhere in the genome

New enzymes often evolve by amplification and divergence of genes encoding enzymes with a weak ability to provide a new function. Experimental studies to date have followed the evolutionary trajectory of an amplified gene, but have not addressed other mutations in the genome when fitness is limited by an evolving gene. We have adapted Escherichia coli in which an enzymes weak secondary activity has been recruited to serve an essential function. While the gene encoding the \"weak-link\" enzyme amplified in all eight populations, mutations improving the new activity occurred in only one. This beneficial allele quickly swept the amplified array, displacing the parental allele. Most adaptive mutations, however, occurred elsewhere in the genome. We have identified the mechanisms by which three of the classes of mutations increase fitness. These mutations may be detrimental once a new enzyme has evolved, and require reversion or compensation, leading to permanent changes in the genome.

evolutionary biology↗

bioRxiv2018

The nuclear interactome of DYRK1A reveals a functional role in DNA damage repair

Loss of function mutations in the protein kinase DYRK1A lead to a syndromic form of autism spectrum disorder and intellectual disability. Conversely, increased DYRK1A dosage is implicated in atypical brain development and neurocognitive deficits in trisomy 21. DYRK1A regulates a diverse array of cellular processes through kinase dependent and independent interactions with substrates and binding partners. Recent evidence implicates DYRK1A in direct regulation of the transcriptional machinery, but many of the molecular details are not yet known. Furthermore, the landscape of DYRK1A interactions in the nucleus is incomplete, impeding progress toward understanding its function in transcription. Here, we used immunoaffinity purification and mass spectrometry to identify nuclear interaction partners of endogenous DYRK1A. These were enriched in DNA damage repair factors, transcriptional elongation factors and E3 ubiquitin ligases. We validated an interaction with RNF169, a factor that promotes homology directed repair upon DNA damage. We further show that knockout of DYRK1A or treatment with DYRK1A inhibitors in HeLa cells impaired efficient recruitment of 53BP1 to DNA double strand breaks induced by ionizing radiation. This nuclear interactome thus reveals a new role for DYRK1A in DNA damage repair and provides a resource for exploring new functions of DYRK1A in the nucleus.

systems biology↗

bioRxiv2018

Cerebral organoid proteomics reveals signatures of dysregulated cortical development associated with human trisomy 21

Human trisomy 21 (Down syndrome) is the most common genetic cause of intellectual disability, and is associated with complex perturbations in protein expression during development. Brain region-specific alterations in neuronal density and composition originate prenatally in trisomy 21 individuals, and are presumed to underlie the intellectual disability and early onset neurodegeneration that characterizes Down syndrome. However, the mechanisms by which chromosome 21 aneuploidy drives alterations in the central nervous system are not well understood, particularly in brain regions that are uniquely human and thus inaccessible to established animal models. Cerebral organoids are pluripotent stem cell derived models of prenatal brain development that have been used to deepen our understanding of the atypical processes associated with human neurobiological disorders, and thus provide a promising avenue to explore the molecular basis for neurodevelopmental alterations in trisomy 21. Here, we employ high-resolution label-free mass spectrometry to map proteomic changes over the course of trisomy 21 cerebral organoid development, and evaluate the proteomic alterations in response to treatment with harmine, a small molecule inhibitor of the chromosome 21 encoded protein kinase DYRK1A. Our results reveal trisomy 21 specific dysregulation of networks associated with neurogenesis, axon guidance and extracellular matrix remodeling. We find significant overlap of these networks show significant overlap with previously identified dysregulated gene expression modules identified in trisomy 21 fetal brain tissue. We show that harmine leads to partial normalization of key regulators of cortical development, including WNT7A and the transcription factors TBR1, BCL11A, and POU3F2, pointing to a causative role for DYRK1A over-expression in neurodevelopmental effects of human trisomy 21.

systems biology↗