An SNRNP70-eGFP knock-in zebrafish line reveals the physiological localisation and dynamics of endogenous SNRNP70 during development

Abstract

SNRNP70 is a core spliceosome RNA-binding protein best known for its essential role in nuclear pre-mRNA splicing. Although traditionally associated with nuclear RNA processing, previous studies have identified important extranuclear functions for SNRNP70 in neurons, including roles in mRNA stability, localisation, and axonal transport. Yet, much of our understanding of SNRNP70 localisation has relied on overexpression or transgenic approaches, leaving a critical gap in our knowledge of where endogenous SNRNP70 resides and how it behaves in living neurons under physiological expression conditions. Here, we address this limitation by establishing and validating a novel zebrafish SNRNP70-eGFP CRISPR knock-in line, enabling direct visualisation of the endogenous protein. eGFP was fused to the C-terminus of endogenous SNRNP70 while retaining the native 3' untranslated region, preserving key regulatory features of the endogenous locus. We demonstrate that the knock-in faithfully reports endogenous SNRNP70 expression and reveals widespread physiological localisation throughout the developing nervous system, including prominent enrichment within axonal and synaptic compartments. Crucially, live in vivo imaging reveals that endogenous SNRNP70 is dynamically localised within neuronal mRNP granules, providing direct evidence of its physiological behaviour in these structures without the confounding effects of protein overexpression. Proximity ligation analyses further demonstrates associations between endogenous SNRNP70 and PABPC1B, FUS, and UPF1, which are established neuronal mRNP granule components. Together, our work provides a validated genetic and imaging resource for investigating SNRNP70 at endogenous levels in the living nervous system. By overcoming key limitations of conventional transgenic and overexpression-based approaches, the SNRNP70-eGFP knock-in enables physiological analysis of SNRNP70 localisation and dynamics and reveals its prominent and dynamic organisation within neuronal mRNP granules. More broadly, this work highlights how endogenous fluorescent tagging can provide a versatile platform for resolving the spatial organisation of RNA-binding proteins in living neurons under physiological expression conditions and provide new insight into the regulation of neuronal mRNA fate.