Biology subjects

Sethi, A. J.

Publications and source records attributed to Sethi, A. J..

5 recordsLinked to original sources

bioRxiv2026

Integrated transcriptomics and proteomics define the TRP channel hierarchy in mouse cortex

Transient receptor potential (TRP) channels are evolutionarily conserved polymodal cation channels that mediate diverse sensory functions across the animal kingdom. Although TRP channels play key roles in peripheral sensation, their expression and functional relevance in the cerebral cortex remain poorly defined. Here, we integrate long- and short-read transcriptomics, targeted qPCR and membrane-aware proteomics to quantify TRP family members in adult mouse cortex. Across transcriptomic platforms, cortical TRP expression is dominated by TRPML, TRPC, and TRPM subfamilies, with lower representation of TRPP/TRPV, whereas Trpa1 and Trpv1 lie near empirical detection thresholds. Our proteomic workflow yields reproducible protein-level evidence for a subset of cortical TRPs, including TRPV2, TRPC4, TRPM3, TRPM7 and TRPP2, consistent with transcript rank order, while TRPA1/TRPV1 do not meet replicate-level protein-group detection criteria under 1% FDR control. Together, these multi-platform measurements establish a quantitative reference for cortical TRP biology and a framework for profiling low-abundance ion channels in complex brain tissue.

molecular biology↗

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

Single-molecule multimodal timing of in vivo mRNA synthesis

mRNA synthesis requires extensive pre-mRNA maturation, the organisation of which remains unclear. Here, we directly sequence pre-mRNA without metabolic labelling or amplification to resolve transcription and multimodal pre-mRNA processing at single-molecule resolution. Using poly(A) tail measurement, we distinguish transcriptionally engaged pre-mRNA from polyadenylated transcripts, revealing that splicing is substantially delayed compared to previous estimates. Splicing is rare for at least 10 kb behind elongating RNA polymerase II such that thousands of genes remain largely unspliced during transcription, revising the notion that splicing is predominantly co-transcriptional. Progressive splicing becomes apparent only on polyadenylated transcripts, suggesting that splicing is commonly activated after 3' end formation. Unexpectedly, we find abundant m6A on unspliced pre-mRNA, indicating that a substantial portion of RNA methylation precedes splicing. This m6A methylation is suppressed around exon boundaries, indicating that m6A topology is established prior to exon junction complex deposition. Finally, we demonstrate that 3' end cleavage occurs multiple kilobases behind transcription in coordination with efficient transcription termination, and further highlight recursive 3' end formation across hundreds of genes. Conserved from human cells to mouse tissues, we illustrate a revised timeline for mRNA synthesis wherein cleavage, termination, and m6A deposition occur earlier than, or at least partially decoupled from, the bulk of splicing, reframing the sequence of early mammalian gene expression.

genomics↗

bioRxiv2022

R2Dtool: Positional interpretation of RNA-centric information in the context of transcriptomic and genomic features

Long-read RNA sequencing enables the mapping of RNA modifications, structures, and protein-interaction sites at the resolution of individual transcript isoforms. To understand the functions of these RNA features, it is critical to analyze them in the context of transcriptomic and genomic annotations, such as open reading frames and splice junctions. To enable this, we have developed R2Dtool, a bioinformatics tool that integrates transcript-mapped information with transcript and genome annotations, allowing for the isoform-resolved analytics and graphical representation of RNA features in their genomic context. We illustrate R2Dtools capability to integrate and expedite RNA feature analysis using epitranscriptomics data. R2Dtool facilitates the comprehensive analysis and interpretation of alternative transcript isoforms. R2Dtool is freely available under the MIT license at: https://github.com/comprna/R2Dtool.

bioinformatics↗

bioRxiv2022

Identification of m6A and m5C RNA modifications at single-molecule resolution from Nanopore sequencing

The epitranscriptome embodies many new and largely unexplored functions of RNA. A significant roadblock hindering progress in epitranscriptomics is the identification of more than one modification in individual transcript molecules. We address this with CHEUI (CH3 (methylation) Estimation Using Ionic current). CHEUI predicts N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual molecules from the same sample, the stoichiometry at transcript reference sites, and differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals to achieve high single-molecule, transcript-site, and stoichiometry accuracies in multiple tests using synthetic RNA standards and cell line data. CHEUIs capability to identify two modification types in the same sample reveals a co-occurrence of m6A and m5C in individual mRNAs in cell line and tissue transcriptomes. CHEUI provides new avenues to discover and study the function of the epitranscriptome.

bioinformatics↗