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<p>T cells are critical effector cells counteracting infections and malignancies. To achieve this, they produce pro-inflammatory cytokines, including IFN-γ and TNF. Cytokine production is a tightly regulated process, but the relative contribution of transcriptional and post-transcriptional regulation to mRNA expression remains unknown. We optimized single-molecule FISH for primary human T cells (T-cell smFISH) to simultaneously quantify nascent RNA, levels of mature mRNA, and its localization with single-cell resolution. T-cell smFISH uncovered heterogeneous cytokine mRNA levels, with high cytokine producers displaying biallelic <i>IFNG</i>/<i>TNF</i> RNA transcription activity. Throughout activation, nuclear cytokine mRNAs accumulated, whereas cytoplasmic cytokine mRNA was degraded through translation-dependent decay. Lastly, T-cell smFISH uncovered cytokine-specific regulation by the RNA-binding protein HuR. Thus, T-cell smFISH provides novel insights in the intricate (post)-transcriptional processes in T cells.</p>

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Single-molecule imaging of transcription dynamics, RNA localization and fate in human T cells

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年10月14日

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  • M Valeria Lattanzio,
  • Nikolina Šoštarić,
  • Nandhini Kanagasabesan,
  • Branka Popović,
  • Antonia Bradarić,
  • Leyma Wardak,
  • Aurélie Guislain,
  • Philipp Savakis,
  • Evelina Tutucci,
  • Monika C Wolkers

摘要

T cells are critical effector cells counteracting infections and malignancies. To achieve this, they produce pro-inflammatory cytokines, including IFN-γ and TNF. Cytokine production is a tightly regulated process, but the relative contribution of transcriptional and post-transcriptional regulation to mRNA expression remains unknown. We optimized single-molecule FISH for primary human T cells (T-cell smFISH) to simultaneously quantify nascent RNA, levels of mature mRNA, and its localization with single-cell resolution. T-cell smFISH uncovered heterogeneous cytokine mRNA levels, with high cytokine producers displaying biallelic IFNG/TNF RNA transcription activity. Throughout activation, nuclear cytokine mRNAs accumulated, whereas cytoplasmic cytokine mRNA was degraded through translation-dependent decay. Lastly, T-cell smFISH uncovered cytokine-specific regulation by the RNA-binding protein HuR. Thus, T-cell smFISH provides novel insights in the intricate (post)-transcriptional processes in T cells.