Pioneer studies have quantified global biases in RNA localization and subcellular differences in translation efficiency that allow local regulation of gene expression (incl. Voigt et al., 2017). Yet most have failed to dissect the molecular mechanisms that target specific transcripts. In the past, this was due to a lack of single-molecule methods as well as to the inherent heterogeneity of RNA localization patterns observed in individual cultured cells in the absence of a clear polarization axis.
To remedy this, we employ clearly polarized organoid model systems that allow us to investigate RNA localization on the single-molecule, single-cell, as well as tissue level - all using a single multi-scale experimental set-up.
To dissect RNA targeting mechanisms and assess how they regulate gene expression locally, we quantify recruitment dynamics, translational efficiency and turnover of individual mRNA transcripts in different subcellular localizations using live single-RNA imaging approaches.
We have established reporter transcripts that are stably expressed in murine intestinal organoid lines engineered to allow live single-particle imaging.
To characterize transport complexes at the molecular level, we assess their mode of transport using single-particle co-localization and diffusion analysis. In addition, we aim to identify the trans-acting factors that recruit RNAs to their target sites through a combination of in-vitro biochemistry approaches.