Biology subjects

McKeating, J.

Publications and source records attributed to McKeating, J..

5 recordsLinked to original sources

bioRxiv2024

Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome

Pseudouridine ({Psi}) is one of the most abundant modifications in cellular RNA. However, its function remains elusive, mainly due to the lack of highly sensitive and accurate detection methods. To address this challenge, we introduced 2-bromoacrylamide-assisted cyclization sequencing (BACS) for quantitative profiling of {Psi} at single-base resolution. Based on novel bromoacrylamide cyclization chemistry, BACS enables a {Psi}-to-C transition. Compared to previous methods, BACS allowed the precise identification of {Psi} positions, especially in densely modified {Psi} regions and consecutive uridine sequences. BACS successfully detected all known {Psi} sites in human rRNA and spliceosomal snRNAs and generated the first quantitative {Psi} map of human snoRNA and tRNA. Furthermore, BACS simultaneously detected adenosine-to-inosine (A-to-I) editing sites and N1-methyladenosine (m1A). Depletion of three key pseudouridine synthases (PUS) enabled us to elucidate the targets and sequence motifs of TRUB1, PUS7, and PUS1 in HeLa cells. We further applied BACS to Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) and identified a highly abundant {Psi}114 site in EBER2. Surprisingly, applying BACS to a panel of RNA viruses demonstrated the absence of {Psi} in their viral transcripts or genomes, shedding light on differences in pseudouridylation between virus families. We anticipate BACS to serve as a powerful tool to uncover the biological importance of {Psi} in future studies.

molecular biology↗

bioRxiv2023

The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome.

Chronic hepatitis B is a global health problem and current treatments only suppress hepatitis B virus (HBV) infection, highlighting the need for new curative treatments. Oxygen levels influence HBV replication and we previously reported that hypoxia inducible factors (HIFs) activate the basal core promoter to transcribe pre-genomic RNA. Application of a probe-enriched long-read sequencing method to map the HBV transcriptome showed an increased abundance of all viral RNAs under low oxygen or hypoxic conditions. Importantly, the hypoxic-associated increase in HBV transcripts was dependent on N6-methyladenosine (m6A) modifications and an m6A DRACH motif in the 5 stem loop of pre-genomic RNA defined transcript half-life under hypoxic conditions. Given the essential role of m6A modifications in the viral transcriptome we assessed the oxygen-dependent expression of RNA demethylases and bio-informatic analysis of published single cell RNA-seq of murine liver showed an increased expression of the RNA demethylase ALKBH5 in the peri-central low oxygen region. In vitro studies with a human hepatocyte derived HepG2 cell line showed increased ALKBH5 gene expression under hypoxic conditions. Silencing the demethylase reduced the levels of HBV pre-genomic RNA and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIF expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen. Author SummaryOxygen levels influence HBV replication and hypoxia inducible factors (HIFs) activate HBV transcription. Long-read sequencing and mapping the HBV transcriptome showed an increased abundance of all viral RNAs under hypoxic conditions that was dependent on N6-methyladenosine modifications. Investigating the oxygen-dependent expression of RNA demethylases identified ALKBH5 as a hypoxic activated gene and silencing its expression showed a key role in regulating HBV and host gene expression under hypoxic conditions.

molecular biology↗

bioRxiv2023

Hypoxia dampens innate immune signalling at early time points and increases Zika virus replication in iPSC-derived macrophages

Type I interferons (IFNs) are the major host defence against viral infection and are induced following activation of cell surface or intracellular pattern recognition receptors, including retinoic-acid-inducible gene I (RIGI)-like receptors (RLRs). All cellular processes are shaped by the microenvironment and one important factor is the local oxygen tension. The majority of published studies on IFN signalling are conducted under atmospheric (18%) oxygen conditions, that do not reflect the physiological oxygen levels in most organs (1-5% O2). We studied the effect of low oxygen on IFN induction and signalling in induced Pluripotent Stem Cell (iPSC)-derived macrophages as a model for tissue-resident macrophages and assessed the consequence for Zika virus (ZIKV) replication. Hypoxic conditions dampened the expression of interferon-stimulated genes (ISGs) following RLR stimulation or IFN treatment at early time points. RNA-sequencing and bio-informatic analysis uncovered several pathways including changes in transcription factor availability, the presence of HIF binding sites in promoter regions, and CpG content that may contribute to the reduced ISG expression. Importantly, hypoxic conditions increased ZIKV replication at early time points, emphasizing the importance of understanding how low oxygen conditions in the local microenvironment affect pathogen sensing and host defence.

microbiology↗

bioRxiv2022

Sleep and circadian rhythm disruption alters the lung transcriptome to predispose to viral infection

Sleep and circadian rhythm disruption (SCRD), as encountered during shift work, increases the risk of respiratory viral infection including SARS-CoV-2. However, the mechanism(s) underpinning higher rates of respiratory viral infection following SCRD remain poorly characterised. To address this, we investigated the effects of acute sleep deprivation on the mouse lung transcriptome. Here we show that sleep deprivation profoundly alters the transcriptional landscape of the lung, causing the suppression of both innate and adaptive immune systems, disrupting the circadian clock, and activating genes implicated in SARS-CoV-2 replication, thereby generating a lung environment that promotes viral infection and associated disease pathogenesis. Our study provides a mechanistic explanation of how SCRD increases the risk of respiratory viral infections including SARS-CoV-2 and highlights therapeutic avenues for the prevention and treatment of COVID-19.

neuroscience↗

bioRxiv2020

Multidrug treatment with nelfinavir and cepharanthine against COVID-19

Antiviral treatments targeting the emerging coronavirus disease 2019 (COVID-19) are urgently required. We screened a panel of already-approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identified two new antiviral agents: the HIV protease inhibitor Nelfinavir and the anti-inflammatory drug Cepharanthine. In silico modeling shows Nelfinavir binds the SARS-CoV-2 main protease consistent with its inhibition of viral replication, whilst Cepharanthine inhibits viral attachment and entry into cells. Consistent with their different modes of action, in vitro assays highlight a synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation. Mathematical modeling in vitro antiviral activity coupled with the known pharmacokinetics for these drugs predicts that Nelfinavir will facilitate viral clearance. Combining Nelfinavir/Cepharanthine enhanced their predicted efficacy to control viral proliferation, to ameliorate both the progression of disease and risk of transmission. In summary, this study identifies a new multidrug combination treatment for COVID-19.

microbiology↗