Regulated microexon alternative splicing in single neurons tunes synaptic function.

Abstract

Microexons are important components of the neuronal transcriptome. Though tiny, their splicing is essential for neuronal development and function. Microexons are typically included in the nervous system and skipped in other tissues, but less is known about whether they are alternatively spliced across neuron types, and if so what the regulatory mechanisms and functional consequences might be. We set out to globally address this question in C. elegans using deep single-cell transcriptomes and in vivo splicing reporters. We find widespread alternative microexon splicing across neuron types. Focusing on a broadly-conserved 9-nucleotide exon in the synaptic vesicle gene unc-13, we find that it is completely skipped in olfactory neurons, but completely included in motor neurons. This splicing pattern is established by two neuronal RNA binding proteins which recruit spliceosomal component PRP-40 to mediate microexon inclusion. Cell-specific microexon alternative splicing is functionally important, as forcing microexon inclusion causes olfactory defects, while forcing microexon skipping causes locomotory defects. These locomotory defects are caused by decreased inhibitory motor neuron synaptic transmission and altered synaptic vesicle distribution. Regulatory features of unc-13 microexon splicing are broadly conserved: related MUN-domain genes in worms, flies, and mice invariably encode microexons, and those we tested are subject to similar regulatory principles (e.g. included in motor neurons, skipped in olfactory neurons, and regulated by the same two RNA binding proteins). Thus, not only is microexon inclusion important for nervous system function, but microexon alternative splicing across neurons is important for tuning neuronal function in individual cell types.