Single-molecule imaging of transcription dynamics, RNA localization and fate in human T cells
M. Valeria Lattanzio (Amsterdam UMC, Sanquin, Oncode Institute)
Nikolina Šoštarić (Sanquin, Oncode Institute, Kavli institute of nanoscience Delft, TU Delft - Applied Sciences, Amsterdam UMC)
Nandhini Kanagasabesan (Oncode Institute, Amsterdam UMC, Sanquin)
Branka Popović (Sanquin, Amsterdam UMC, Oncode Institute)
Antonia Bradarić (Amsterdam UMC, Oncode Institute, Sanquin)
Leyma Wardak (Sanquin, Amsterdam UMC, Oncode Institute)
Aurélie Guislain (Amsterdam UMC, Sanquin, Oncode Institute)
Philipp Savakis (Vrije Universiteit Amsterdam)
Monika C. Wolkers (Oncode Institute, Sanquin, Amsterdam UMC)
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Abstract
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.
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