Splicing in a single neuron is coordinately controlled by RNA binding proteins and transcription factors.

Abstract

Single-cell transcriptomes are established by transcription factors (TFs), which determine a cell's gene-expression complement. Post-transcriptional regulation of single-cell transcriptomes, and the RNA binding proteins (RBPs) responsible, are more technically challenging to determine, and combinatorial TF-RBP coordination of single-cell transcriptomes remains unexplored. We used fluorescent reporters to visualize alternative splicing in single <i>Caenorhabditis elegans</i> neurons, identifying complex splicing patterns in the neuronal kinase <i>sad-1</i>. Most neurons express both isoforms, but the ALM mechanosensory neuron expresses only the exon-included isoform, while its developmental sister cell the BDU neuron expresses only the exon-skipped isoform. A cascade of three cell-specific TFs and two RBPs are combinatorially required for <i>sad-1</i> exon inclusion. Mechanistically, TFs combinatorially ensure expression of RBPs, which interact with <i>sad-1</i> pre-mRNA. Thus a combinatorial TF-RBP code controls single-neuron <i>sad-1</i> splicing. Additionally, we find 'phenotypic convergence,' previously observed for TFs, also applies to RBPs: different RBP combinations generate similar splicing outcomes in different neurons.