Robust single-molecule tracking of RNA abundance and dynamics via TSSOT.

Single-RNA imaging in living cells offers precise insights into the spatiotemporal regulation of gene expression. However, achieving stable, efficient, and reproducible RNA labeling remains challenging. To address this, we developed TSSOT (tandem split-mNeonGreen-based signal-optimized mRNA tracking), a robust live-cell imaging system that ensures consistent RNA labeling across multiple cell passages and provides highly reproducible, quantitative measurements at single-molecule resolution within individual cells. Leveraging TSSOT, we assessed the efficacy of various gene manipulation techniques and identified critical factors influencing Cas13d-mediated RNA cleavage. TSSOT further demonstrated that downregulation of mRNA export factors NXF1 and ALYREF leads to profound nuclear mRNA export defects. Furthermore, we employed TSSOT to spatiotemporally resolve the dynamic redistribution of mRNAs during stress granule formation. By providing precise quantification of RNA abundance, spatial organization, and temporal behavior, TSSOT is a valuable tool for live-cell RNA analysis, contributing to a better understanding of the dynamic RNA landscape.