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

## TL;DR

This study uses a new imaging technique to explore how T cells regulate cytokine mRNA expression at the single-cell level.

## Contribution

The study introduces an optimized single-molecule FISH workflow for primary human T cells to study transcription and post-transcriptional regulation.

## Key findings

- T-cell smFISH reveals heterogeneous cytokine mRNA levels with biallelic transcription in high producers.
- Nuclear cytokine mRNAs accumulate during activation, while cytoplasmic mRNAs are degraded translation-dependently.
- The RNA-binding protein HuR regulates cytokine mRNA specifically, affecting polyadenylation and nuclear export.

## 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.

T cells produce pro-inflammatory cytokines like IFN-γ and TNF. This study presents an optimized single-molecule FISH workflow in primary human T cells for studying how (post)-transcriptional regulation shapes cytokine mRNA expression.

Heterogenic T cell responses stem from mono/biallelic transcription and single/double cytokine mRNA expression.Mature mRNA accumulates within the nucleus in activated T cells.IFNG and TNF –but not IL2– mRNA undergoes translation-dependent decay.HuR deficiency uncouples mRNA from protein expression levels.HuR acts transcript-specifically, mediating polyadenylation of TNF, and possibly nuclear export of IFNG.

Heterogenic T cell responses stem from mono/biallelic transcription and single/double cytokine mRNA expression.

Mature mRNA accumulates within the nucleus in activated T cells.

IFNG and TNF –but not IL2– mRNA undergoes translation-dependent decay.

HuR deficiency uncouples mRNA from protein expression levels.

HuR acts transcript-specifically, mediating polyadenylation of TNF, and possibly nuclear export of IFNG.

A novel, optimized T-cell smFISH workflow allows to simultaneously quantify nascent and mature mRNA, and its localization with single-cell resolution.