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Significant Variations in Double-Stranded RNA Levels in Cultured Skin Cells

Lookup NU author(s): Dr Dr Shaymaa Sadeq, Dr Suwalak Chitcharoen, Dr Surar Al-Hashimi, Ann Rattanaburi, John CasementORCiD, Dr Andreas Werner

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Abstract

© 2024 by the authors.Endogenous double-stranded RNA has emerged as a potent stimulator of innate immunity. Under physiological conditions, endogenous dsRNA is maintained in the cell nucleus or the mitochondria; however, if protective mechanisms are breached, it leaches into the cytoplasm and triggers immune signaling pathways. Ectopic activation of innate immune pathways is associated with various diseases and senescence and can trigger apoptosis. Hereby, the level of cytoplasmic dsRNA is crucial. We have enriched dsRNA from two melanoma cell lines and primary dermal fibroblasts, including a competing probe, and analyzed the dsRNA transcriptome using RNA sequencing. There was a striking difference in read counts between the cell lines and the primary cells, and the effect was confirmed by northern blotting and immunocytochemistry. Both mitochondria (10–20%) and nuclear transcription (80–90%) contributed significantly to the dsRNA transcriptome. The mitochondrial contribution was lower in the cancer cells compared to fibroblasts. The expression of different transposable element families was comparable, suggesting a general up-regulation of transposable element expression rather than stimulation of a specific sub-family. Sequencing of the input control revealed minor differences in dsRNA processing pathways with an upregulation of oligoadenylate synthase and RNP125 that negatively regulates the dsRNA sensors RIG1 and MDA5. Moreover, RT-qPCR, Western blotting, and immunocytochemistry confirmed the relatively minor adaptations to the hugely different dsRNA levels. As a consequence, these transformed cell lines are potentially less tolerant to interventions that increase the formation of endogenous dsRNA.


Publication metadata

Author(s): Sadeq S, Chitcharoen S, Al-Hashimi S, Rattanaburi S, Casement J, Werner A

Publication type: Article

Publication status: Published

Journal: Cells

Year: 2024

Volume: 13

Issue: 3

Online publication date: 25/01/2024

Acceptance date: 22/01/2024

Date deposited: 26/02/2024

ISSN (electronic): 2073-4409

Publisher: Multidisciplinary Digital Publishing Institute (MDPI)

URL: https://doi.org/10.3390/cells13030226

DOI: 10.3390/cells13030226

Data Access Statement: RNAseq data generated and discussed in this project have been submitted to SRA (accession number PRJNA1053654).

PubMed id: 38334619


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