Biology subjects

Sudbery, I. M.

Publications and source records attributed to Sudbery, I. M..

9 recordsLinked to original sources

bioRxiv2026

DNA Damage Driven Viability Loss and Transcriptional Reprogramming in Chinese Hamster Ovary Cell Perfusion Culture

Intensified perfusion cultures promise higher yields and consistent product quality, yet extended runs frequently stall due to declining Chinese hamster ovary (CHO) cell viability at high cell density. Here, we identify DNA damage accumulation as a central, previously underappreciated driver of this limitation. Using two antibody-expressing CHO cell lines operated at a control and high cell density in perfusion bioreactors, we combined time-resolved transcriptomics with molecular, biophysical and super-resolution imaging analyses. We observed a progressive, global downregulation of DNA damage response (DDR) pathways accompanied by a time-dependent accumulation of DNA lesions. Notably, {gamma}H2AX signalling declined despite DNA damage, indicating impaired damage sensing and repair. Concomitantly, RNA polymerase II protein levels and transcriptional hub organisation were markedly reduced, consistent with widespread transcriptional dysfunction preceding loss of viability. An extended 21-day perfusion run confirmed continued viability decline beyond day 14, supporting a cumulative damage model. Comparison with HEK293 cells revealed substantially more efficient DNA repair, underscoring an intrinsically attenuated DDR in CHO cells. Together, these findings demonstrate that unrepaired DNA damage fundamentally constrains perfusion culture longevity, exposing limits of CHO genomic plasticity and highlighting DDR pathways as promising targets for host cell engineering to enhance perfusion performance.

bioengineering↗

bioRxiv2024

Widespread 3' UTR splicing regulates expression of oncogene transcripts in sequence-dependent and independent manners

BackgroundSplicing in 3 untranslated regions (3 UTRs) is generally considered a signal to elicit transcript degradation via nonsense-mediated decay (NMD) due to the presence of an exon junction complex (EJC) downstream of the stop codon. However, 3 UTR intron (3UI)-containing transcripts are widespread and highly expressed in both normal tissues and cancers. ResultsHere we present and characterise a novel transcriptome assembly built from 7897 solid tumour and normal samples from The Cancer Genome Atlas. We identify thousands of 3UI-containing transcript isoforms, many of which are expressed across multiple cancer types. We find that the expression of core NMD component UPF1 negatively correlates with global 3UI splicing between normal samples, however this correlation is lost in colon cancer. We find that 3UIs found exclusively within 3 UTRs (bona-fide 3UIs) are not predominantly NMD-sensitising, unlike introns present in 3 UTRs due to premature termination. We identify HRAS as an example where 3UI splicing rescues the transcript from NMD. Bona-fide, but not premature termination codon (PTC) carrying 3UI-transcripts are spliced more in cancer samples compared to matched normals in the majority of cancer types analysed. In colon cancer, differentially spliced 3UI-containing transcripts are enriched in the canonical Wnt signalling pathway, with CTNNB1 being the most over-spliced in colon cancer compared to normal. We show that manipulating Wnt signalling can further regulate splicing of Wnt component transcript 3 UTRs. ConclusionsOur results indicate that 3 UTR splicing is not a rare occurrence, especially in colon cancer, where 3 UTR splicing regulates transcript expression in EJC-dependent and independent manners.

genomics↗

bioRxiv2023

N6-methyladenosine modification is not a general trait of viral RNA genomes

Despite the nuclear localization of the m6A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m6A-modified. However, these findings are mostly based on m6A-seq, an antibody-dependent technique with a high rate of false positives. Here, we addressed the presence of m6A in CHIKV and DENV RNAs. For this, we combined m6A-seq and the antibody-independent SELECT and nanopore direct RNA sequencing techniques with functional, molecular, and mutagenesis studies. Following this comprehensive analysis, we found no evidence of m6A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m6A machinery did not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection had no effect on the m6A machinerys localization. Our results challenge the prevailing notion that m6A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches.

microbiology↗

bioRxiv2023

Ageing-related defects in macrophage function are driven by MYC and USF1 transcriptional programmes

Macrophages are central innate immune cells whose function declines with age. The molecular mechanisms underlying age-related immunity changes remain poorly understood, particularly in human macrophages. We report a substantial reduction in phagocytosis, migration and chemotaxis in human monocyte-derived macrophages (MDMs) from older (>50 years) compared with younger (18-30 years) donors, alongside downregulation of transcription factors MYC and USF1 with age. In MDMs from young donors, knockdown of MYC or USF1 decreased phagocytosis and chemotaxis and altered expression of genes associated with these functions, as well as adhesion and extracellular matrix remodelling. A concordant dysregulation of MYC and USF1 target genes was also seen in MDMs from older donors. Furthermore, older age and loss of either MYC or USF1 in MDMs led to an increased cell size, altered morphology and reduced actin content. Together, these results define MYC and USF1 as key drivers of MDM age-related functional decline and identify downstream targets to improve macrophage function in ageing.

immunology↗

bioRxiv2023

hnRNPUL1 ensures efficient Integrator-mediated cleavage of snRNAs and is mutated in amyotrophic lateral sclerosis

Integrator cleaves nascent RNA, triggering RNA polymerase II transcription termination, but how cleavage is regulated is poorly understood. Here we show hnRNPUL1 ensures efficient Integrator-mediated cleavage of nascent RNA downstream of snRNA genes and, in the case of U2 snRNA, binds a terminal stem-loop involved in this process. In the nucleoplasm, hnRNPUL1 binds U4 snRNA and SART3 and enables efficient reformation of the U4:U6 di-snRNP for further rounds of pre-mRNA splicing. Sustained hnRNPUL1 loss leads to reduced levels of snRNAs, defects in histone mRNA 3' end processing and loss of Cajal bodies. hnRNPUL1 binds RNA through multiple domains, including a globular central domain comprising tightly juxtaposed SPRY and dead polynucleotide kinase folds. This latter fold allows binding to 5'-monophosphorylated RNAs in a mutually exclusive manner with ATP binding and functions as an XRN2 antagonist when overexpressed. We identify a cohort of amyotrophic lateral sclerosis patients harbouring disruptive mutations in hnRNPUL1. SMN loss in spinal muscular atrophy and hnRNPUL1 loss both disrupt snRNP biogenesis, leading to motor neuron death, suggesting a common aetiology.

molecular biology↗

bioRxiv2020

Over-accessible chromatin links myeloma initiating genetic events to oncogenic transcriptomes and aberrant transcription factor regulatory networks

Multiple myeloma is a genetically heterogeneous cancer of the bone marrow plasma cells (PC). Myeloma initiating genetic events define subgroups (MIE) and drive distinct oncogenic transcriptomes that converge into a mutually exclusive overexpression of CCND1 and CCND2 oncogenes. Here, with reference to normal PC, we dissect how MIE impact the chromatin regulatory landscape of MM. We find that chromatin accessibility combined with transcriptome profiling classifies myeloma genetic subgroups, while in a topologically constrained manner, distal rather than proximal regulatory elements influence myeloma transcriptomes. Across and within MIE-defined subgroups, genes and pathways critical for myeloma biology can be linked to developmentally activated or de novo formed enhancers. We show that existing transcription factors, co-opted to organise highly ordered, aberrant regulatory networks, generate known and novel myeloma cell dependencies and help identify prognostic markers. Finally, we discover and functionally validate the critical enhancer that regulates ectopic expression of CCND2 in MM.

cancer biology↗

bioRxiv2019

CGAT-core: a python framework for building scalable, reproducible computational biology workflows

In the genomics era computational biologists regularly need to process, analyse and integrate large and complex biomedical datasets. Analysis inevitably involves multiple dependent steps, resulting in complex pipelines or workflows, often with several branches. Large data volumes mean that processing needs to be quick and efficient and scientific rigour requires that analysis be consistent and fully reproducible. We have developed CGAT-core, a python package for the rapid construction of complex computational workflows. CGAT-core seamlessly handles parallelisation across high performance computing clusters, integration of Conda environments, full parameterisation, database integration and logging. To illustrate our workflow framework, we present a pipeline for the analysis of RNAseq data using pseudoalignment.\n\nAvailabilityCGAT-core is freely available under an MIT licence for installation and use, including source code at https://github.com/cgat-developers/cgat-core\n\nContactandreas.heger@imm.ox.ac.uk (AH), david.sims@imm.ox.ac.uk (DS), adam.cribbs@imm.ox.ac.uk (AC)\n\nSupplementary information:

genomics↗

bioRxiv2018

A proteome-wide screen reveals widespread roles for the phosphatase Cdc14 in the Candida albicans cell cycle

The chromosome complement of the human fungal pathogen Candida albicans is unusually unstable, suggesting that process of nuclear division is error prone. The Cdc14 phosphatase plays a key role in organising the intricate choreography of mitosis and cell division. In order to understand the role of Cdc14 in C. albicans we used quantitative proteomics to identify proteins that physically interact with CaCdc14. To distinguish genuine Cdc14-interactors from proteins that bound non-specifically bound to the affinity matrix we used an orthogonal approach of a substrate trapping mutant combined with mass spectrometry analysis using stable isotope labelling in cell culture (SILAC). The results identified 126 proteins that interact with Cdc14 of which 80% are novel. In this set, 53 proteins play known roles in the cell regulating the attachment of the mitotic spindle to kinetochores, mitotic exit, cytokinesis, licensing of DNA replication by re-activating pre-replication complexes, and DNA repair. Five Cdc14-interacting proteins with previously unknown functions localized to the Spindle Pole Bodies (SPBs). Intriguingly, 83 proteins that only interacted with Cdc14 in yeast were significantly enriched in components of the ergosterol biosynthesis pathway targeted by azole anti-fungal drugs. Thus we have greatly expanded the set of known substrates of this key cell cycle regulator in C. albicans.\n\nAuthor summaryCandida albicans is an important fungal pathogen causing life-threatening bloodstream infections in humans, as well as debilitating mucosal infections. Here we used Mass Spectroscopy to identify proteins that physically interacted with an enzyme called Cdc14. By removing phosphate groups from proteins, and thus regulating their function, this enzyme orchestrates the intricate molecular mechanisms of nuclear division to ensure that each daughter cell receives a full complement of chromosomes. C. albicans is unusual in the way that changes in chromosome number and composition are much more common than in other organisms. This suggests that the process of nuclear division may be more error prone in C. albicans. Our work identified 126 proteins that physically associate with Cdc14 and are thus potential substrates, including 53 proteins that we know are involved in many cell cycle processes that are necessary for nuclear and cell division. Thus, we have laid the ground work to study how changes in chromosomal composition may arise due to errors in nuclear division in this important pathogen. Unexpectedly, we found that Cdc14 may also act on proteins involved in the synthesis of ergosterol, a key lipid in the cell membrane. Azoles, a major class of antifungal drugs, inhibit the synthesis of ergosterol, so Cdc14 may also be involved in the action of azoles and thus one possible way in which drug resistance arises.

cell biology↗

bioRxiv2018

Co-transcriptional loading of RNA export factors shapes the human transcriptome.

During gene expression, RNA export factors are mainly known for driving nucleocytoplasmic transport. Whilst early studies suggested that the Exon Junction Complex provides a binding platform for them, subsequent work proposed that they are only recruited by the Cap-Binding Complex to the 5 end of RNAs, as part of TREX. Using iCLIP, we show that the export receptor Nxf1 and two TREX subunits, Alyref and Chtop, are actually recruited to the whole mRNA co-transcriptionally via splicing but before 3-end processing. Consequently, Alyref alters splicing decisions and Chtop regulates alternative polyadenylation. Alyref is recruited to the 5-end of RNAs by CBC and our data reveal subsequent binding to RNAs near EJCs. We demonstrate eIF4A3 stimulates Alyref deposition not only on spliced RNAs close to EJC sites, but also single exon transcripts. Our study reveals mechanistic insights into the co-transcriptional recruitment of mRNA export factors and how this shapes the human transcriptome.

molecular biology↗