There exists considerable heterogeneity in the transcriptomes of individual cells within any given population1,2. This variation in transcriptomic profiles is now known to result from mRNA homeostasis mechanisms, such as size-scaling, where transcript counts increase proportionally with cell size to maintain concentration homeostasis1,3,4. Contemporary research has highlighted how cells adapt mRNA synthesis rates to facilitate size scaling, and modify both synthesis and degradation rates in response to perturbations to either end of the RNA lifecycle—a process known as mRNA buffering3,5,6.
While these mechanisms likely involve multiple pathways, how cells dynamically regulate transcription at the single-gene level remains largely unknown. Our current knowledge of how these changes are facilitated are derived from studies modelling transcription on fixed cell data, or employing inducible systems that do not recapitulate endogenous expression patterns3,6–8.
As such, this project interrogates how cells regulate transcription at the single gene level to facilitate mRNA homeostasis as a function of cell-cycle and cell-size. We use CRISPR-Cas9 gene editing at endogenous loci to achieve visualisation of transcription with high spatial and temporal sensitivity with lattice lightsheet microscopy, in combination with single molecule RNA-FISH (smRNA-FISH) to capture static snapshots of mRNA abundance for individual genes. Integrating these approaches, we aim to unify our current knowledge of these mechanisms and synthesise a cohesive framework for understanding the timing and nature of transcriptional adaptations in living cells.
We demonstrate in our data, the conserved cell-size scaling phenomena at POLR2A in both live and fixed cell data, with and without perturbations, to reveal how the parameters governing gene expression can shift to facilitate a mRNA concentration homeostasis.