Single molecule imaging of transcription dynamics, RNA localization and fate in T cells
T cells are critical effector cells against infections and malignancies. To achieve this, they produce pro-inflammatory cytokines, including IFN-{gamma} and TNF. Cytokine production is a tightly regulated process. The relative contribution of transcriptional and post-transcriptional regulation to mRNA expression is, however, unknown. We therefore optimized single-molecule FISH for primary human T cells (T-cell smFISH) to simultaneously quantify nascent RNA, mature mRNA levels 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.