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Long-read RNA sequencing reveals an isoform switching pat-2 tern in healthy microglial cell lines exposed to radiotherapy

ABSTRACT: Radiotherapy (RT) is a fundamental treatment for brain tumors, but often leads to long-term neurotoxicity, partly caused by microglial dysfunction. In this study, we used long-read RNA sequencing (LR-RNA-seq) to map the isoform-specific transcriptome of human microglial HMC3 cells after RT exposure. HMC3 human microglial cell line was treated with standard radiotherapy and, after RNA extraction and library preparation, LR-RNA-seq was performed. Further bioinformatic analyses allowed to investigate on the isoform switching pattern after radiotherapy exposure. Gene-level analysis found 591 differentially expressed genes, with positive enrichment of PI3K/AKT-related pathways and negative enrichment of translation-associated processes. Isoform-level analysis uncovered 827 significant isoform switching events across 420 genes, often involving shifts from protein-coding transcripts to non-coding variants. Functional annotation showed that most isoform switches resulted in the loss of open reading frames and protein domains, especially in ribosomal proteins and translation machinery components. Exploratory proteomic analysis identified 191 differentially abundant proteins, including a significant reduction in RPL7A, providing partial protein level support for translation-associated transcriptomic remodeling.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Mixed Disorder As Reaction To Stress

SUBMITTER: Francesco Bellia

LAB HEAD: Francesco Bellia

PROVIDER: PXD081768 | Pride | 2026-08-06

REPOSITORIES: Pride

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Project description:Transcription and translation are intertwined processes where mRNA isoforms are crucial intermediaries. However, methodological limitations in analyzing translation at the mRNA isoform level have impaired our ability to comprehensively establish links between the full-length transcripts and the translatome. This has left gaps in our understanding of critical biological processes, regulatory mechanisms, and disease progression. To address this, we develop an integrated computational and experimental framework called long-read Ribo-STAMP (LR-Ribo-STAMP). LR-Ribo-STAMP capitalizes on advancements in long-read sequencing and RNA-base editing-mediated technologies to simultaneously and scalably profile translation and transcription at both gene and mRNA isoform levels for the first time. In this report, we show agreement between gene-level translation profiles obtained with LR-Ribo-STAMP and those from previously validated short-read Ribo-STAMP data in unperturbed cells. At the mRNA isoform level, we show that LR-Ribo-STAMP successfully profiles translation in unperturbed cells and links mRNA isoforms and regulatory features, such as upstream ORFs (uORFs) and regulatory sequences, to translation measurements. We further demonstrate the method’s effectiveness in profiling disease models by profiling translation at gene and isoform levels in a triple-negative breast cancer cell line under normoxia and hypoxia. Here, we find that LR-Ribo-STAMP effectively delineates orthogonal transcriptional and translation shifts between conditions at gene and isoform levels. At the isoform level, LR-Ribo-STAMP uniquely identifies key regulatory elements and shifts in mRNA isoform transcription that correlate with changes in translational, providing an example of insight that can inform the development of novel therapeutics. Overall, LR-Ribo-STAMP is a significant advancement in translation methods and can have profound implications for basic research and clinical applications.

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Long-read RNA sequencing reveals an isoform switching pat-2 tern in healthy microglial cell lines exposed to radiotherapy

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