Allele-specific RNA imaging shows that allelic imbalances can arise in tissues through transcriptional bursting

Extensive cell-to-cell variation exists even among putatively identical cells, and there is great interest in understanding how the properties of transcription relate to this heterogeneity. Differential expression from the two gene copies in diploid cells could potentially contribute, yet our ability to measure from which gene copy individual RNAs originated remains limited, particularly in the context of tissues. Here, we demonstrate quantitative, single molecule allele-specific RNA FISH adapted for use on tissue sections, allowing us to determine the chromosome of origin of individual RNA molecules in formaldehyde-fixed tissues. We used this method to visualize the allele-specific expression of Xist and multiple autosomal genes in mouse kidney. By combining these data with mathematical modeling, we evaluated models for allele-specific heterogeneity, in particular demonstrating that apparent expression from only one of the alleles in single cells can arise as a consequence of low-level mRNA abundance and transcriptional bursting.Author summary: In mammals, most cells of the body contain two genetic datasets: one from the mother and one from the father, and—in theory—these two sets of information could contribute equally to produce the molecules in a given cell. In practice, however, this is not always the case, which can have dramatic implications for many traits, including visible features (such as fur color) and even disease outcomes. However, it remains difficult to measure the parental origin of individual molecules in a given cell and thus to assess what processes lead to an imbalance of the maternal and paternal contribution. We adapted a microscopy technique—called allele-specific single molecule RNA FISH—that uses a combination of fluorescent tags to specifically label one type of molecule, RNA, depending on its origin, for use in mouse kidney sections. Focusing on RNAs that were previously reported to show imbalance, we performed measurements and combined these with mathematical modeling to quantify imbalance in tissues and explain how these can arise. We found that we could recapitulate the observed imbalances using models that only take into account the random processes that produce RNA, without the need to invoke special regulatory mechanisms to create unequal contributions.