Multimodal measurements of 3’UTR-mediated control of neuronal mRNAs reveal links between gene regulatory mechanisms
2Department of Biosystems Science and Engineering, ETH Zurich, Switzerland
Massively parallel reporter assays (MPRAs) generate controlled measurements of the regulatory activity of a DNA or RNA sequence. Here, we applied novel MPRAs to map the regulatory landscape of 3’UTRs in neurons. Neurons use a particularly large repertoire of regulatory mechanisms to fine-tune expression of their genes and 3’ untranslated regions (3’UTRs) are known hubs for regulation of RNA localization, stability and translation. We developed three MPRAs and functionally tested 50,000 native or designed 3’UTR sequences to identify regions that (a) trigger localization of the mRNA to neurites, (b) mediate increased RNA stability or degradation and (c) control the amount of protein made.
We identify 3’UTR regions regulating specifically one of the measured outputs (localization, stability or translation control). In addition, we also find 3’UTR elements affecting more than one aspect of gene expression. Such sequences can be involved in the coordination of different gene regulatory layers through utilizing the same trans-acting factors. Integrating MPRA measurements of subcellular RNA localization and turnover suggests that reduced stability of an mRNA entails its restriction to the cell body, but that increased stability is not sufficient to explain its accumulation in neuronal processes. RNA localization elements often also functioned as translational repressors, hinting at a common regulatory machinery mediating transport of RNAs to neuronal processes and keeping them translationally repressed on the way.
Computational models trained on our data were able to predict the localization behavior of native mRNAs, suggesting that the rules uncovered by our MPRA also apply to native full-length transcripts.