β-actin mRNA Interactome Mapping in Mouse Embryonic Stem Cells (mESCs) and mESC-Derived Neurons by RNA-Proximity Labeling

Abstract

Die Dissertation ist gesperrt bis zum 15. Oktober 2026 !

In many cell types, messenger RNAs (mRNAs) are transported to specific subcellular locations, where they are translated, creating localized protein pools with diverse functions. The encoded proteins themselves can regulate temporal/spatial gene expression and translation, and are generally required for maintaining cellular asymmetry, proper embryonic development or diverse neuronal functions. A classic example of a localized mRNA is β-actin mRNA, which is highly abundant in dendrites and enriched in the growth cone of neurons. The localization and subsequent translation of β-actin mRNA into proteins in growth cones is crucial for axon guidance and synaptic plasticity, processes that are vital to learning and memory. The basic components necessary for mRNA localization have been identified: the mRNA itself, the translational machinery with its regulatory elements, and a cellular delivery system. However, the mechanisms orchestrating mRNA localization and local translation remain to be elucidated. In this thesis, I aimed to unravel the components of the β-actin mRNA granules in mouse embryonic stem cells (mESCs) and mESC-derived neurons using an RNA-BioID approach. Adopting two CRISPR/Cas9 approaches, firstly a 24x MS2-β-actin mRNA cell line was generated, and then a biotin ligase fusion protein, called ‘MicroID’, was stably integrated into the murine Hipp11 safe-harbor locus. This enabled the MicroID fusion protein to be expressed and active in both mESCs and mESC-derived neurons. Additionally, I verified that the 24x MS2 array does not disrupt β-actin mRNA transcription, translation or its localization. Applying RNA-BioID to these engineered cell lines revealed both known (e.g. IGF2BP1) and unknown (MEX3A, YTHDF3) β-actin mRNA-associated proteins. The precise roles of MEX3A and YTHDF3 in neuronal β-actin mRNA localization warrant further investigation. This work underscores the complexity of an mRNA’s journey and the ongoing need to identify the molecular mechanisms and regulatory elements involved during an mRNA’s localization process. Further characterization of MEX3A and YTHDF3 functions promises to move us toward a more nuanced understanding of the sophisticated regulatory networks governing sitespecific protein synthesis in neurons.