Biology subjects

Weatheritt, R. J.

Publications and source records attributed to Weatheritt, R. J..

8 recordsLinked to original sources

bioRxiv2025

SWARM: A Single-Molecule Workflow for High-Precision Profiling of RNA Modifications

Nanopore direct RNA sequencing promises to decode the epitranscriptome by detecting multiple modifications on individual RNA molecules, but its potential for biological discovery is hampered by high false-positive rates. We present SWARM, an AI-based framework designed to overcome this fundamental limitation. Its key innovation is a crosstalk-aware training strategy that incorporates non-target modifications and orthogonally validated cellular signals, enabling high-precision detection of m6A, pseudouridine ({Psi}), and m5C at single-nucleotide and single-molecule resolution. Using rigorous in vitro and cellular RNA benchmarks, SWARM outperforms existing tools and maintains strong agreement with orthogonal methods. Applying SWARM across mammalian tissues reveals thousands of novel modification sites with confirmed motifs and localisation patterns. Our high-resolution multi-tissue modification map revealed no evidence of widespread m6A-{Psi} interplay in predominant writer contexts, challenging models of a coordinated epitranscriptomic code. We further discovered a previously unrecognised splicing-shaped mode of {Psi} deposition, whereby TRUB1-mediated pseudouridylation preferentially occurs after exon-exon ligation, consistent with local RNA structure stabilisation. SWARM provides a robust, universally applicable tool for epitranscriptome discovery.

bioinformatics↗

bioRxiv2025

High-content imaging reveals the ability of microexons to shape protein localisation

Alternative splicing provides a pervasive means to expand proteome complexity, yet how it reorganises protein interactions and constrains where proteins act within cell remains unclear. By constructing an interface-resolved interaction network composed of 17,660 experimentally defined contact sites, we reveal that tissue-specific alternative splicing remodels protein connectivity by reshaping modular protein architecture. Longer exons reshape local interaction patterns whereas microexons fine-tune key interfaces linking distinct cellular processes. Integration of subcellular localisation data further indicates that such rewiring can redistribute proteins within cells. To test this, we developed a high-content imaging approach to systematically evaluate the influence of individual exons on protein localisation and screened a targeted library of protein isoforms differing in individual exons. 38% of the tested isoform pairs altered localisation, with microexons, although typically shorter than five amino acids, accounting for a substantial proportion of these effects. Bioinformatic and structural analyses identified that microexons can extend secondary structural regions and reposition charged residues, suggesting a potential to modulate local electrostatic environments. Consistent with this, biochemical analysis of a four-amino acid microexon in sorting nexin 2 - identified through our screen - confirmed that residue insertion, rather than side chain chemistry, was driving differences in protein localisation through repositioning of a flanking, charged residue. Together, these findings describe a principle by which alternative splicing fine-tunes interface architecture to coordinate protein assembly, localisation, and proteome organisation.

molecular biology↗

bioRxiv2025

Isoform-specific single-cell perturb-seq reveals distinct functions of alternative promoters in drug response

CRISPR interference (CRISPRi) screens have emerged as powerful tools for dissecting gene function, yet their application to genes with multiple promoters, which comprise over 60% of human genes, remains poorly understood. Here, we demonstrate that CRISPR-dCas9-based screens exhibit widespread promoter specificity, with untargeted promoters often showing compensatory upregulation to maintain gene expression. Leveraging this selective targeting of individual promoters within the same gene, we developed isoform-specific single-cell Perturb-Seq to systematically analyse alternative promoter function. Our analysis revealed that alternative promoters in 48.3% of targeted genes drive distinct transcriptional programs. This suggests that promoter selection represents a fundamental mechanism for generating cellular diversity rather than mere transcriptional redundancy. In breast cancer models, this promoter-specific targeting revealed differential effects on drug sensitivity, where distinct estrogen receptor (ESR1) promoters showed opposing influences on tamoxifen response and patient survival. These findings demonstrate the necessity of promoter-level analysis in functional genomics and suggest new strategies for therapeutic intervention through promoter-specific targeting.

genomics↗

bioRxiv2024

High-Accuracy RNA Integrity Definition for Unbiased Transcriptome Comparisons with INDEGRA

RNA sample integrity variability introduces biases and obscures natural RNA degradation, posing a significant challenge in transcriptomics. To address this, we developed the Direct Transcriptome Integrity (DTI) measure, a universal and robust RNA integrity metric based on nanopore sequencing. By accurately modeling RNA fragmentation, DTI provides a reliable assessment of sample quality. Integrated into the INDEGRA package (freely available at https://github.com/Arnaroo/INDEGRA), we provide tools to correct false discoveries and enable precise differential expression and RNA degradation analyses, even for challenging sample types. INDEGRA software can be used to accurately measure RNA DTI stability metric, isolate biological component of RNA degradation from technical biases, compare biological RNA stability transcriptome-wide and suppress false degradation-induced differential gene expression hits to allow broad comparisons across samples of different quality DTI offers a straightforward and accurate method for assessing RNA degradation, characterizing both overall sample integrity and transcript-specific degradation rates using direct RNA sequencing (DRS) data. Calculated through INDEGRA, DTI reveals inter- and intra-transcript variability in degradation, while INDEGRA separates RNA degradation from mapping inaccuracies, and connects degradation profiles to RNA fragmentation rates. By leveraging INDEGRA, researchers can minimize false differential transcript abundance findings caused by variations in overall sample integrity, while preserving genuine transcript-specific differences in stability and degradation. INDEGRA supports integration with widely used differential transcript abundance tools like DESeq2, limma-voom, and edgeR, enabling seamless analysis pipelines. INDEGRA enhances the accuracy and reliability of RNA quantification in high-throughput data and simplifies comparisons across diverse transcriptomic datasets, including those derived from different tissues, species, or experimental protocols.

bioinformatics↗

bioRxiv2024

The conserved landscape of RNA modifications and transcript diversity across mammalian evolution

Gene expression programs underpin the development of shared phenotypes, yet the importance of transcript complexity in shaping mammalian evolution remains unclear. Here we present a comprehensive long-read direct RNA sequencing atlas of full-length transcripts and their m6A modifications across six tissues (hippocampus, frontal cortex, cerebellum, testes, skeletal muscle, and liver) in five mammals (human, mouse, rat, dog, and cow) and a non-mammalian out-group (chicken). Our analysis reveals that 29% of genes have multiple mammalian-conserved alternative transcripts, with 31% of these genes showing tissue-specific switching of the major transcript isoforms. We uncover extensive conservation of coordinated splicing events, primarily driven by tissue-specific mutually associated exon splicing, particularly in neural tissues and cytoskeletal genes. At the epitranscriptome level, we find that 14.2% of m6A RNA modifications are conserved across mammals, with 39% of analysed genes containing a conserved m6A RNA modification. Our work provides unprecedented insight into the evolution of transcript complexity and the epitranscriptome, highlighting their potential roles in shaping mammalian phenotypic diversity and providing a valuable resource for understanding post-transcriptional regulation across mammalian evolutionary time.

genomics↗

bioRxiv2021

Active DNA demethylation of developmental cis-regulatory regions predates vertebrate origins

DNA methylation (5-methylcytosine; 5mC) is a repressive gene-regulatory mark required for vertebrate embryogenesis. Genomic 5mC is tightly regulated through the coordinated action of DNA methyltransferases, which deposit 5mC, and TET enzymes, which participate in its active removal through the formation of 5-hydroxymethylcytosine (5hmC). TET enzymes are essential for mammalian gastrulation and activation of vertebrate developmental enhancers, however, to date, a clear picture of 5hmC function, abundance, and genomic distribution in non-vertebrate lineages is lacking. By employing base-resolution 5mC and 5hmC quantification during sea urchin and lancelet embryogenesis, we shed light on the roles of non-vertebrate 5hmC and TET enzymes. We find that these invertebrate deuterostomes employ TET enzymes for targeted demethylation of regulatory regions associated with developmental genes and show that the complement of identified 5hmC-regulated genes is conserved to vertebrates. This work thus demonstrates that active 5mC removal from regulatory regions is a common feature of deuterostome embryogenesis suggestive of unexpected deep conservation of a major gene-regulatory module.

genomics↗

bioRxiv2021

Evolutionary dynamics of circular RNAs in primates

Many primate genes produce non-coding circular RNAs (circRNAs). However, the extent of circRNA conservation between closely related species remains unclear. By comparing tissue-specific transcriptomes across over 70 million years of primate evolution, we identify that within 3 million years circRNA expression profiles diverged such that they are more related to species identity than organ type. However, our analysis also revealed a subset of circRNAs with conserved neural expression across tens of millions of years of evolution. These circRNAs are defined by an extended downstream intron that has shown dramatic lengthening during evolution due to the insertion of novel retrotransposons. Our work provides comparative analyses of the mechanisms promoting circRNAs to generate increased transcriptomic complexity in primates.

genomics↗

bioRxiv2017

Whippet: an efficient method for the detection and quantification of alternative splicing reveals extensive transcriptomic complexity

Alternative splicing (AS) is a widespread process underlying the generation of transcriptomic and proteomic diversity in metazoans. Major challenges in comprehensively detecting and quantifying patterns of AS are that RNA-seq datasets are expanding near exponentially, while existing analysis tools are computationally inefficient and ineffective at handling complex splicing patterns. Here, we describe Whippet, a method that rapidly, and with minimal hardware requirements, models and quantifies splicing events of any complexity without significant loss of accuracy. Using an entropic measure of splicing complexity, Whippet reveals that approximately 33% of human protein coding genes contain complex AS events that result in substantial expression of multiple splice isoforms. These events frequently affect tandem arrays of folded protein domains. Remarkably, high-entropy AS events are more prevalent in tumour relative to matched normal tissues, and these differences correlate with increased expression of proto-oncogenic splicing factors. Whippet thus affords the rapid and accurate analysis of AS events of any complexity, and as such will facilitate biomedical research.

genomics↗