Graphical Abstract Highlights d Image-based quantification of mRNA and protein abundance in thousands of single cells d Variability in mRNA-protein ratios is determined by cellular state and microenvironment d Cellular state and microenvironment influence JUN gene expression at multiple levels d Variability in cytoplasmic patterning of JUN mRNA depends on the microenvironment SUMMARYA long-standing question in quantitative biology is the relationship between mRNA and protein levels of the same gene. Here, we measured mRNA and protein abundance, the phenotypic state, and the population context in thousands of single human cells for 23 genes by combining a unique collection of cell lines with fluorescently tagged endogenous genomic loci and quantitative immunofluorescence with branched DNA single-molecule fluorescence in situ hybridization and computer vision. mRNA and protein abundance displayed a mean singlecell correlation of 0.732 at steady state. Single-cell outliers of linear correlations are in a specific phenotypic state or population context. This is particularly relevant for interpreting mRNA-protein relationships during acute gene induction and turnover, revealing a specific adaptation of gene expression at multiple steps in single cells. Together, we show that singlecell protein abundance can be predicted by multivariate information that integrates mRNA level with the phenotypic state and microenvironment of a particular cell.
AUTHOR CONTRIBUTIONSL.P. and D.P. conceived the project; B.K., M.K., and J.E. created and provided endogenously tagged HeLa cell lines; D.P. performed all experiments and analyzed all data; L.P. and D.P. wrote the paper.