A Fluorescence in Situ Hybridization Method To Quantify mRNA Translation by Visualizing Ribosome–mRNA Interactions in Single Cells

Single-molecule fluorescence
in situ hybridization (smFISH) is
a simple and widely used method to measure mRNA transcript abundance
and localization in single cells. A comparable single-molecule in
situ method to measure mRNA translation would enable a more complete
understanding of gene regulation. Here we describe a fluorescence assay to detect ribosome interactions with mRNA (FLARIM). The method adapts smFISH to visualize and characterize
translation of single molecules of mRNA in fixed cells. To visualize
ribosome–mRNA interactions, we use pairs of oligonucleotide
probes that bind separately to ribosomes (via rRNA) and to the mRNA
of interest, and that produce strong fluorescence signals via the
hybridization chain reaction (HCR) when the probes are in close proximity.
FLARIM does not require genetic manipulation, is applicable to practically
any endogenous mRNA transcript, and provides both spatial and temporal
information. We demonstrate that FLARIM is sensitive to changes in
ribosome association with mRNA upon inhibition of global translation
with puromycin. We also show that FLARIM detects changes in ribosome
association with an mRNA whose translation is upregulated in response
to increased concentrations of iron.