Visualizing mRNA Translation in situ in Single Cells

Citation

Burke, Kelly Suzanne (2018) Visualizing mRNA Translation in situ in Single Cells. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z92B8W6R. https://resolver.caltech.edu/CaltechTHESIS:07292017-201142545

Abstract

Translation of mRNA is tightly regulated in cells to ensure that proteins are synthesized at the right time, in the right location, and at appropriate levels. Single-molecule fluorescence in situ hybridization (smFISH) is a simple and widely used method to measure mRNA transcription through determining the abundance and localization of mRNAs in single cells. A comparable single-molecule in situ method to measure mRNA translation would enable a more complete understanding of gene regulation. In this thesis, we describe the development and characterization of 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 ribosomal RNA) 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 first characterize FLARIM in mouse fibroblast cells. We show 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. Finally, we demonstrate FLARIM in mouse hippocampal neurons, in which local translation of mRNA in the neuronal processes is essential to cell growth and development. We first demonstrate FLARIM in neurons using probes for β-actin mRNA. We then show that FLARIM detects increased transcription and translation of activity regulated cytoskeletal protein (Arc) mRNA in response to neuronal activation with brain-derived neurotrophic factor (BDNF). We also compare the cellular distribution between the major microtubule-associated protein 2 (MAP2) mRNA isoforms, whose translation will be characterized in future FLARIM experiments. Overall, this work expands the capability of smFISH to study mRNA translation in addition to transcription, and it shows the utility of in situ translation analysis in characterizing single-cell gene expression.

Thesis Files