Visualizing mRNA quality control: Mechanistic insights from translational dynamics

The genetic information in the DNA of a cell is expressed via transcription of DNA into mRNA and translation of the mRNA into protein. Tight regulation of translation is important to ensure that genes are expressed at the correct level and that no errors are introduced during gene expression. In this thesis, I develop several methods to visualize in real-time the translation of single mRNA molecules and their degradation by mRNA quality control. The basis of these methods is the SunTag translation visualization system, in which nascent peptides are labelled by fluorescent antibodies as soon as they are produced by the ribosome. This labelling results in bright fluorescent spots that are detectable by fluorescence microscopy at sites in a cell were an mRNA of interest is translated. The brightness of the fluorescent spot can be used as a readout for the amount of ribosomes that are currently translating. Using this method, translation of individual mRNA molecules can be quantitatively assessed and followed in real-time for prolonged periods of time. We then expand on this method by combing detection of translation with detection of mRNA degradation that happens as a consequence of mRNA quality control mechanisms such as nonsense-mediated mRNA decay and non-stop decay. Finally, this thesis also described the first steps in the development of a new procedure for COVID-19 diagnostic testing in which most manual steps of the testing procedure are performed by a custom-build robot that can handle up to 20.000 samples per day.