Biology subjects

Harrison, P. F.

Publications and source records attributed to Harrison, P. F..

8 recordsLinked to original sources

bioRxiv2025

ADAR1p150 RNA binding, independent of A-to-I RNA editing, buffers immunogenicity from the tonic type I IFN induced transcriptome in vivo.

ADAR1 edits adenosine to inosine (A-to-I) in double-stranded RNA to prevent MDA5 sensing of cellular transcripts, its key physiological role. However, editing-independent functions of ADAR1 remain poorly understood. Using a series of Adar1 mutant mice rescued by loss of MDA5 and PKR, we investigated isoform-specific, editing-independent roles of ADAR1 in vivo. We found that the cytoplasmic ADAR1p150 isoform is essential for maintaining peripheral T cell numbers and differentiation of hematopoietic stem and progenitor cells (HSPCs). In bone marrow transplants, ADAR1p150 protein, but not its editing activity, was crucial for T cell regeneration and HSPC repopulation, demonstrating a cell-intrinsic function in hematopoiesis. Experiments with IFN{beta}-treatment of purified HSPCs in vitro and in vivo IFNAR1 neutralization revealed hypersensitivity to tonic type I interferon (IFN) in the absence of ADAR1p150. Using cell lines, we demonstrate that type I IFN activates the OAS-RNaseL pathway, leading to cell death. This study shows that tonic type I IFN induces immunogenic cellular RNAs in sterile conditions. ADAR1p150 suppresses immune sensing of self-dsRNAs through both editing-dependent (MDA5) and editing-independent (PKR, OAS-RNaseL) mechanisms. Thus, ADAR1p150 protein levels and activity combine to set the threshold for tolerance to self-derived dsRNAs.

immunology↗

bioRxiv2023

Primary human myoblasts display only minor alternative polyadenylation compared to the transformed C2C12 model of muscle differentiation

Alternative polyadenylation has been linked to multiple developmental and disease transitions. The prevailing hypothesis being that differentiated cells use longer 3 UTRs with expended regulatory capacity whereas undifferentiated cells use shorter 3 UTRs. Here, we describe the gene expression and alternative polyadenylation profiles of human primary myoblasts over a time course of differentiation. Contrary to expectations, only minor changes in the 3 end choice were observed. To reconcile this finding with published research, we devised a new bioinformatic method to compare the degree of alternative polyadenylation in the differentiation of primary human and immortalized murine (C2C12) myoblasts. Differentiated human primary myotubes display only half the alternative polyadenylation of the mouse model, with less than 1/10 of the genes undergoing alternative polyadenylation in C2C12 cells showing evidence of alternative processing in human primary muscle differentiation. A global reduction in the expression of cleavage and polyadenylation factors in C2C12, but not in primary human myotubes may explain the lack of alternative polyadenylation in this system. Looking more broadly at transcriptome changes across differentiation shows that less than half of the genes differentially expressed in the immortalized model were recapitulated in primary cells. Of these, important metabolic pathways, such as glycolysis and sterol biosynthesis, showed divergent regulation. Collectively, our data caution against using immortalized cell lines, which may not fully recapitulate human muscle development, and suggest that alternative polyadenylation in the differentiation of primary cells might be less pronounced than previously thought.

molecular biology↗

bioRxiv2023

Active maintenance of CD8+ T cell naivety through regulation of global genome architecture

The differentiation of naive CD8+ cytotoxic T lymphocytes (CTLs) into effector and memory states results in large scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organisation reflect or underpin these transcriptional programs. We utilised Hi-C to map changes in the spatial organisation of long-range genome contacts within naive, effector and memory virus-specific CD8+ T cells. We observed that the architecture of the naive CD8+ T cell genome was distinct from effector and memory genome configurations with extensive changes within discrete functional chromatin domains. However, deletion of the BACH2 or SATB1 transcription factors was sufficient to remodel the naive chromatin architecture and engage transcriptional programs characteristic of differentiated cells. This suggests that the chromatin architecture within naive CD8+ T cells is preconfigured to undergo autonomous remodelling upon activation, with key transcription factors restraining differentiation by actively enforcing the unique naive chromatin state. One Sentence SummaryCD8+ T cell naivety is actively maintained by transcription factors that enforce a distinct, naive chromatin architecture. HighlightsO_LICD8+ T cell differentiation states are underscored by distinct chromatin looping architectures. C_LIO_LIChromatin loops juxtapose CTL state appropriate enhancers, transcription factors and genes. C_LIO_LIEffector and memory CTLs have similar genome architectures, explaining rapid memory recall. C_LIO_LICTL differentiation is restrained by BACH2 and SATB1, which enforce a naive loop architecture. C_LI

immunology↗

bioRxiv2022

langevitour: smooth interactive touring of high dimensions, demonstrated with scRNA-Seq data

langevitour displays interactive animated 2D projections of high-dimensional datasets. Langevin Dynamics is used to produce a smooth path of projections. Projections are initially explored at random. A "guide" can be activated to look for an informative projection, or variables can be manually positioned. After a projection of particular interest has been found, continuing small motions provide a channel of visual information not present in a static scatter-plot. langevitour is implemented in Javascript, allowing for a high frame rate and responsive interaction, and can be used directly from the R environment or embedded in HTML documents produced using R. The widget is demonstrated using single-cell RNA sequencing (scRNA-Seq) data. langevitours linear projections provide a less distorted view of this data than commonly used non-linear dimensionality reductions such as UMAP.

bioinformatics↗

bioRxiv2020

Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast

Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3 untranslated region in order to regulate mRNA function. Here we present a systematic analysis of 3 end formation factors, which revealed 3UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3UTRs. We show that the anti-cancer drug cordycepin, 3 deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an rpb1 mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3 end processing and controlled by the availability of 3 end factors, nucleotide levels and chromatin landscape.

cell biology↗

bioRxiv2020

Splice-site Strength Estimation: A simple yet powerful approach to analyse RNA splicing

RNA splicing, and variations in this process referred to as alternative splicing, are critical aspects of gene regulation in eukaryotes. From environmental responses in plants to being a primary link between genetic variation and disease in humans, splicing differences confer extensive phenotypic changes across diverse organisms1-3. Current approaches for analysing splicing rely on quantifying variant transcripts (i.e., isoforms) or splicing events (i.e., intron retention, exon skipping etc)4, 5. However, regulation of splicing occurs at the level of selection of individual splice sites, which results in variation in the abundance of isoforms and/or splicing events. Here, we present a simple approach to quantify the strength of individual splice sites, which determines their selection in a splicing reaction. Splice-site strength, as a quantitative phenotype, allows us to analyse splicing precisely in unprecedented ways. We demonstrate the power of this approach in defining the genomic determinants of the strength of individual splice-sites through GWAS. Our pilot-GWAS with more than thousand splice sites hints that cis-sequence divergence and competition between splice-sites and are among the primary determinants of variation in splicing among natural accessions of Arabidopsis thaliana. This approach allows deciphering the principles of splicing, which in turn has implications that range from agriculture to medicine.

genetics↗

bioRxiv2018

Widespread cytoplasmic polyadenylation programs asymmetry in the germline and early embryo

BACKGROUNDThe program of embryonic development is launched by selective activation of a silent maternal transcriptome. In Caenorhabditis elegans, nuclei of the adult germline are responsible for the synthesis of at least two distinct mRNA populations; those required for housekeeping functions, and those that program the oocyte-to-embryo transition. We mapped this separation by changes to the length-distribution of poly(A)-tails that depend on GLD-2 mediated cytoplasmic polyadenylation and its regulators genome-wide.\n\nRESULTSMore than 1000 targets of cytoplasmic polyadenylation were identified by differential polyadenylation. Amongst mRNA with the greatest dependence on GLD-2 were those encoding RNA binding proteins with known roles in spatiotemporal patterning such as mex-5 and pos-1. In General, the 3 UTR of GLD-2 targets were longer, contained cytosine-patches, and were enriched for non-standard polyadenylation-motifs. To identify the deadenylase that initiated transcript silencing, we depleted the known deadenylases in the gld-2(0) mutant background. Only the loss of CCF-1 suppressed the short-tailed phenotype of GLD-2 targets suggesting that in addition to its general role in RNA turnover, this is the major deadenylase for regulatory silencing of maternal mRNA. Analysis of poly(A)-tail length-change in the embryo lacking specific RNA-binding proteins revealed new candidates for asymmetric expression in the first embryonic divisions.\n\nCONCLUSIONThe concerted action of RNA binding proteins exquisitely regulates GLD-2 activity in space and time. We present our data as interactive web resources for a model where GLD-2 mediated cytoplasmic polyadenylation regulates target mRNA at each stage of worm germline and early embryonic development.

developmental biology↗

bioRxiv2018

Topconfects: a package for confident effect sizes in differential expression analysis provides improved usability ranking genes of interest

BackgroundA differential gene expression analysis may produce a set of significantly differentially expressed genes that is too large to easily investigate, so that a means of ranking genes by their biological interest level is desirable. The life-sciences have grappled with the abuse of p-values to rank genes for this purpose. As an alternative, a lower confidence bound on the magnitude of Log Fold Change (LFC) could be used to rank genes, but it has been unclear how to reconcile this with the need to perform False Discovery Rate (FDR) correction. The TREAT test of McCarthy and Smyth is a step in this direction, finding genes significantly exceeding a specified LFC threshold. Here we describe the use of test inversion on TREAT to present genes ranked by a confidence bound on the LFC, while still controlling FDR.\n\nResultsTesting the Topconfects R package with simulated gene expression data shows the method outperforming current statistical approaches across a wide range of experiment sizes in the identification of genes with largest LFCs. Applying the method to a TCGA breast cancer data-set shows the method ranks some genes with large LFC higher than would traditional ranking by p-value. Importantly these two ranking methods lead to a different biological emphasis, in terms both of specific highly ranked genes and gene-set enrichment.\n\nConclusionsThe choice of ranking method in differential expression analysis can affect the biological interpretation. The common default of ranking by p-value is implicitly by an effect size in which each gene is standardized to its own variability, rather than comparing genes on a common scale, which may not be appropriate. The Topconfects approach of presenting genes ranked by confident LFC effect size is a variation on the TREAT method with improved usability, removing the need to fine-tune a threshold parameter and removing the temptation to abuse p-values as a de-facto effect size.

bioinformatics↗