Abstract
BackgroundAlternative RNA processing plays an essential role in shaping cell identity and connectivity in the central nervous system. 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 describe 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.ResultsWe developed a means of undertaking motoneuron-specific CLIP to explore motoneuron-specific protein–RNA interactions relative to studies of the whole spinal cord in mouse. This allowed us to pinpoint differential RNA regulation specific to motoneurons, revealing a major role for NOVA in regulating cytoskeleton interactions in motoneurons. In particular, NOVA specifically promotes the palmitoylated isoform of the cytoskeleton protein Septin 8 in motoneurons, 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.
Highlights
Alternative RNA processing plays an essential role in shaping cell identity and connectivity in the central nervous system
Using crosslinking and immunoprecipitation (CLIP), a method that allows stringent purification of protein–RNA complexes captured in vivo, we identified NOVA targets in mouse neocortex [16,17,18,19], and have estimated that NOVA participates in the regulation of ~ 7% of brain-specific alternative splicing events in mouse neocortex [20]
NIH/3T3 cells ectopically expressing NOVA2 or AcGFP-NOVA2 were subjected to HITS-CLIP using antibodies against NOVA and GFP, respectively (Additional file 1: Supplemental material and methods; Additional file 2: Figure S1A)
Summary
Alternative RNA processing plays an essential role in shaping cell identity and connectivity in the central nervous system. This is believed to involve differential regulation of RNA processing in various cell types. Recent technological breakthroughs using RiboTag and BAC-TRAP mouse lines have allowed for translational profiling at single cell type resolution [10,11,12,13]. These studies revealed remarkable differences in the population of translating mRNAs across various CNS cell types, highlighting the degree of molecular heterogeneity among neuronal cells. We develop a complementary and more general means to study RNA processing and regulation in a cell type-specific manner
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