A single molecule approach reveals uncoordinated expression of genes
Gandhi, Saumil J.
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The expression of functionally related genes, such as those encoding essential subunits of multi-protein complexes, is thought to be co-regulated to ensure proper stoichiometry. But the evidence supporting this view comes from ensemble measurements that detect population averages, which may be significantly different from what is occurring in individual cells. In fact, it has been shown that stochastic fluctuations in mRNA levels often generate considerable differences in gene expression between individual cells, even if they are genetically identical. Although proteins comprising essential complexes or pathways have similar abundances and lower variability between cells, it is not known whether this co-regulation occurs at the level of transcription, mRNA abundance, or protein expression. Therefore, coordination in the expression of genes must be studied with direct measurements of mRNA abundance within individual cells.;We describe here the development of a novel fluorescence in situ hybridization based approach for measuring correlations between the expression of various genes by counting the number of single mRNAs within Saccharomyces cerevisiae cells. Combination of single mRNA FISH with automated image analysis algorithms made it possible to measure correlations in the expression of genes in hundreds of single cells with unprecedented sensitivity and precision.;We hypothesized that mRNA abundances of essential genes encoding proteins in the same complex or pathway would have a higher correlation coefficient than transcripts of functionally unrelated genes. Our results revealed that transcripts of temporally induced genes are highly correlated in individual cells. But in contrast, transcription of constitutive genes encoding essential subunits of multi-protein complexes is not coordinated due to stochastic fluctuations. Even more remarkably, transcript levels of two endogenous alleles of the same gene within the same cell are also uncorrelated. These results suggest a fundamental shift in the way we must think about coordination of various biological processes within a cell. Transcription of functionally related genes responsible for performing specific biological tasks is not coordinated within individual cells. Rather, genes are clueless entities that produce mRNAs randomly, irrespective of the necessary concentrations of the final gene product. Therefore, the regulation of precise stoichiometry must occur post-transcriptionally, and likely post-translationally.