Spatiotemporally resolved transcriptomics reveals subcellular RNA kinetic landscape

Spatiotemporal regulation of the cellular transcriptome is crucial for proper protein expression<br> and cellular function. However, the intricate subcellular dynamics of RNA synthesis, decay,<br> export, and translocation remain obscured due to the limitations of existing transcriptomics<br> methods Here, we report a spatiotemporally resolved RNA mapping method (TEMPOmap) to<br> uncover subcellular RNA profiles across time and space at the single-cell level in heterogeneous<br> cell populations. TEMPOmap integrates pulse-chase metabolic labeling of the transcriptome with<br> highly multiplexed three-dimensional (3D) in situ sequencing to simultaneously profile the age<br> and location of individual RNA molecules. Using TEMPOmap, we constructed the subcellular<br> RNA kinetic landscape of 991 genes in human HeLa cells from upstream transcription to<br> downstream subcellular translocation. Clustering analysis of critical RNA kinetic parameters<br> across single cells revealed kinetic gene clusters whose expression patterns were shaped by multistep<br> kinetic sculpting. Importantly, these kinetic gene clusters are functionally segregated,<br> suggesting that subcellular RNA kinetics are differentially regulated to serve molecular and<br> cellular functions in a cell-cycle-dependent manner. We further demonstrated that functionallysegregated<br> RNA kinetics could be seen in heterogeneous human primary cell cultures, revealing<br> cell-type-dependent RNA dynamic regulation. Together, these single-cell spatiotemporally<br> resolved transcriptomics measurements provide us the gateway to uncovering new gene regulation<br> principles and understanding how kinetic strategies enable precise RNA expression in time and<br> space.