Extranuclear activities of SNRNP70 in zebrafish neurons

Abstract

This work investigates the cytoplasmic role of the RNA-binding protein SNRNP70 in zebrafish motor neuron development, transcriptomic regulation, and protein-protein networks implicated with messenger RNA transport and metabolism in neurons. Using an overexpression model, this work demonstrates that cytoplasmic SNRNP70 negatively impacts motor neuron morphology, resulting in shortened axonal projections and reduced dendritic arbour complexity. These morphological alterations are accompanied by transcriptomic changes which suggest deficits in mitochondrial function, energy and iron metabolism, and lipid homeostasis. This occurs concomitantly to the alternative splicing of genes governing axon guidance and dendritic branching. Altogether, this suggests that the overexpression of the cytoplasmic SNRNP70 protein impairs neuronal growth by reducing energy availability, destabilising membranes, and limiting synaptic plasticity.

In parallel to the transcriptomic characterisation of the cytoplasmic SNRNP70 protein, a novel CRISPR knock-in zebrafish line was generated, in which the endogenous C-terminal of the protein was tagged with eGFP. This tool greatly facilitated the sub-cellular visualisation of the protein and revealed its localisation throughout the central nervous system, including retinal and motor neuron-rich regions implicated in neurodegenerative disease.

In concert with this, proteomics approaches involving immunoprecipitation mass spectrometry, co-immunoprecipitation and the proximity ligation assay shed light upon the protein interactome of SNRNP70 within the nervous system. The SNRNP70-eGFP KI tool was used extensively throughout and provided considerable opportunities for visualising the protein interactome of SNRNP70 in vivo and in vitro. On this basis, the proteomic analysis further established SNRNP70’s associations with the snRNP biogenesis machinery, members of the Sm ring complex, and major neuronal trans-port proteins (e.g. FUS, FMR1, IGF2BP1, kif20b), highlighting its role within neuronal granules and mRNP complexes. Furthermore, the colocalisation studies within zebrafish embryos revealed potentially significant associations between SNRNP70 and proteins involved in neurodevelopmental (FMR1) and neurodegenerative disease (FUS), pointing to potential contributions of these associations to disease pathobiology.

Overall, this thesis expands the understanding of the cytoplasmic SNRNP70 protein and provides evidence that the protein is involved in the regulation of neuronal morphology and provides proteomics data to support SNRNP70 being considered a neuronal RNA granule protein. Beyond this, the research tool developed with the CRISPR-KI

greatly enhances the research capable of being conducted into the protein and represents a considerable step forward in characterising its role in the cytoplasm.

Date of Award	20 May 2026
Original language	English
Awarding Institution	University of Bath
Supervisor	Nicholas Nikolaou (Supervisor), Shobbir Hussain (Supervisor) & Keith Vance (Supervisor)