Biology subjects

Overholt, K. J.

Publications and source records attributed to Overholt, K. J..

3 recordsLinked to original sources

bioRxiv2024

Nucleolar accumulation of APE1 through condensates is mediated by rRNA forming G-quadruplex structures

APE1 (apurinic/apyrimidinic endodeoxyribonuclease 1) is the main endonuclease of the base excision repair (BER) pathway acting on abasic (AP)-sites in damaged DNA. APE1 is an abundant nuclear protein with a higher concentration than other BER pathway enzymes, and therefore, improper expression and localization of this factor could lead to the accumulation of toxic DNA intermediates. Altered APE1 sub-cellular localization, expression levels, or hyper-acetylation are associated with cancer development suggesting the importance of a fine-tuning mechanism for APE1 nuclear-associated processes. Recent work highlighted multi-functional roles of APE1, including rRNA quality control. However, how rRNA influences the sub-cellular localization and activity of APE1 remains poorly understood, but previously underappreciated APE1-RNA interactions may influence the ability of this protein to form biomolecular condensates and tune APE1 partitioning into nucleoli. Since nucleolar accumulation of ectopic proteins could be the result of overexpression strategies, it is imperative to have cellular models to study APE1 trafficking under physiological conditions. Here we created the first cell line to express fluorescently tagged APE1 at its endogenous locus, enabling live-cell imaging. Live-cell imaging demonstrates that APE1 nucleolar accumulation requires active rRNA transcription. When modeled in vitro, APE1 condensate formation depends on RNA G-quadruplex (rG4) structures in rRNA and is modulated by critical lysine residues of APE1. This study sheds light on the mechanisms underlying APE1 trafficking to the nucleolus and formation of RNA-dependent APE1 nucleolar condensates that may modulate a switch between the activity of this factor in rRNA processing and DNA damage repair. Significance StatementWe created and characterized the first endogenous, fluorescently tagged cell line to study APE1 subcellular trafficking under physiological and stress conditions. Using this cell line, we show that APE1 nucleolar enrichment occurs under physiological conditions and, performing in vitro droplet assays, we associate APE1 condensates with active transcription of RNA G-quadruplexes, abundantly present in healthy nucleoli. This work deepens our understanding of APE1s role in healthy cells in the absence of DNA damage and provide a novel mechanism for how this protein responds to stress. Our results suggest that phase separation is an important part of how DNA damage repair proteins switch between their normal physiological functions and their ability to correct DNA lesions.

molecular biology↗

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↗

bioRxiv2020

Dissecting the common and compartment-specific features of COVID-19 severity in the lung and periphery with single-cell resolution

As the global COVID-19 pandemic continues to escalate, no effective treatment has yet been developed for the severe respiratory complications of this disease. This may be due in large part to the unclear immunopathological basis for the development of immune dysregulation and acute respiratory distress syndrome (ARDS) in severe and critical patients. Specifically, it remains unknown whether the immunological features of the disease that have been identified so far are compartment-specific responses or general features of COVID-19. Additionally, readily detectable biological markers correlated with strata of disease severity that could be used to triage patients and inform treatment options have not yet been identified. Here, we leveraged publicly available single-cell RNA sequencing data to elucidate the common and compartment-specific immunological features of clinically severe COVID-19. We identified a number of transcriptional programs that are altered across the spectrum of disease severity, few of which are common between the lung and peripheral immune environments. In the lung, comparing severe and moderate patients revealed severity-specific responses of enhanced interferon, A20/I{kappa}B, IL-2, and IL-6 pathway signatures along with broad signaling activity of IFNG, SPP1, CCL3, CCL8, and IL18 across cell types. These signatures contrasted with features unique to ARDS observed in the blood compartment, which included depletion of interferon and A20/I{kappa}B signatures and a lack of IL-6 response. The cell surface marker S1PR1 was strongly upregulated in patients diagnosed with ARDS compared to non-ARDS patients in {gamma}{delta} T cells of the blood compartment, and we nominate S1PR1 as a potential marker for immunophenotyping ARDS in COVID-19 patients using flow cytometry. HIGHLIGHTSO_LICOVID-19 disease severity is associated with a number of compositional shifts in the cellular makeup of the blood and lung environments. C_LIO_LITranscriptional data suggest differentially expressed cell surface proteins as markers for COVID-19 immunophenotyping from BALF and PBMC samples. C_LIO_LISeverity-specific features COVID-19 manifest at the pathway level, suggesting distinct changes to epithelia and differences between local and systemic immune dynamics. C_LIO_LIImmune-epithelial cellular communication analysis identifies ligands implicated in transcriptional regulation of proto-oncogenes in the lung epithelia of severe COVID-19 patients. C_LIO_LINetwork analysis suggests broadly-acting dysregulatory ligands in the pulmonary microenvironment as candidate therapeutic targets for the treatment of severe COVID-19. C_LI

immunology↗