Cell type-specific CLIP reveals that NOVA regulates cytoskeleton interactions in motoneurons

Abstract

BackgroundAlternative RNA processing plays an essential role in shaping cell identity and connectivity in the central nervous system (CNS). This is believed to involve differential regulation of RNA processing in various cell types. However, in vivo study of cell-type specific post-transcriptional regulation has been a challenge. Here, we developed a sensitive and stringent method combining genetics and CLIP (crosslinking and immunoprecipitation) to globally identify regulatory interactions between NOVA and RNA in the mouse spinal cord motoneurons (MNs).ResultsWe developed a means of undertaking MN-specific CLIP to explore MN-specific protein-RNA interactions relative to studies of the whole spinal cord. This allowed us to pinpoint differential RNA regulation specific to MNs, revealing major role for NOVA in regulating cytoskeleton interactions in MNs. In particular, NOVA specifically promotes the palmitoylated isoform of a cytoskeleton protein Septin 8 in MNs, which enhances dendritic arborization.ConclusionsOur study demonstrates that cell type-specific RNA regulation is important for fine-tuning motoneuron physiology, and highlights the value of defining RNA processing regulation at single cell type resolution.

Nova proteins direct synaptic integration of somatostatin interneurons through activity-dependent alternative splicing

“…Yang et al, 2016). Previous HITS-CLIP analysis has revealed that Nova1 and Nova2 share an almost identical RNA-binding domain (YCAY) (Licatalosi et al, 2008;Ule et al, 2006;2003b;Yuan et al, 2018a). Strikingly, the Nova-binding motif was found to be significantly enriched within activity-dependent targets and at a higher frequency than other neuronal splice factors (i.e.…”

“…To address whether Nova1 and Nova2 differentially affect connectivity and maturation, we asked what AS networks they control within SST cINs during development. Given that they share a very similar RNA-binding motif and are found associated with one another in vivo, they were thought to function cooperatively (Licatalosi et al, 2008;Racca et al, 2010;Yuan et al, 2018b). However recently, it has been shown that in addition to their synergistic roles, Nova1 and Nova2 proteins each control distinct AS gene networks (Saito et al, 2019;2016).…”

“…However recently, it has been shown that in addition to their synergistic roles, Nova1 and Nova2 proteins each control distinct AS gene networks (Saito et al, 2019;2016). We thus chose to examine changes in AS within SST cINs in Nova1, Nova2 or Nova1/2 compound conditional knockout mice (Saito et al, 2019;Yuan et al, 2018b). Using FAC sorting, we isolated SST cINs from SST Cre ;Nova1 F/F or SST Cre ; ;Nova2 F/F or SST Cre ;Nova1 F/F ; Nova2 F/F mice on an Ai9 reporter background (referred to henceforth at SST-Nova1, SST-Nova2 and SST-dKO, respectively) at P8 (Figure 4A).…”

Approaches for measuring the dynamics of RNA–protein interactions

“…Beyond modifications that have made CLIP protocols more efficient (Van Nostrand et al, 2017;Zarnegar et al, 2016), CLIP has also been combined with powerful genetic tools to provide new perspectives on protein-RNA interactions and alternatively processed mRNAs in specific cell populations in different tissues (H. W. Hwang et al, 2017;Saito et al, 2019;Yuan et al, 2018). For example, cTag-CLIP (conditionally tagged) utilizes transgenic mice and Cre-lox technology to express GFP-tagged versions of the RBP NOVA2 in specific cell types depending on the promoter used to drive expression of the Cre recombinase (H. W. Hwang et al, 2017;Jereb et al, 2018;Saito et al, 2019;Ule et al, 2018).…”

“…The technique is particularly suited for tissues with high cellular heterogenity, like brain. Using this approach, comparison of NOVA2-RNA interactions in motoneurons versus whole spinal cord revealed motoneuron-specific RNA regulatory functions of NOVA2 that promote branching of dendrites (Yuan et al, 2018). Similarly, NOVA2-cTag-CLIP allowed resolution of functional differences in NOVA2-mediated alternative splicing of the same transcripts in either excitatory or inhibitory neurons (Saito et al, 2019).…”