Concentration-dependent Splicing through Suboptimal Motifs Enables Waves of Gene Regulation in Neuronal Development

Abstract

Alternative splicing, which occurs in over 95% of human genes, is the process by which exons are differentially included in transcripts produced from the same gene to produce a variety of transcript isoforms. This mode of post-transcriptional gene regulation endows the 20,000 protein-coding genes in the human genome with tunable expression, manifold protein–protein interactions, and an additional layer by which to regulate protein localization, activity, and signal response. Increasingly, alternative splicing is understood to be a significant contributor to organismal complexity in animals.

The Rbfox family of splicing factors regulates alternative splicing during animal development and in disease, impacting thousands of exons in the maturing brain, heart, and muscle. Although it is well established that Rbfox binds to the RNA sequence GCAUG with high affinity and specificity, this motif is responsible for only half of the Rbfox binding sites observed in cellular and neuronal contexts. We incubated recombinant RBFOX2 with over 60,000 mouse and human transcriptomic sequences to reveal substantial binding to several moderate-affinity, non-GCAYG sites at a physiologically relevant range of RBFOX2 concentrations. We find that these “secondary motifs” bind Rbfox robustly in cells and that several together can exert regulation comparable to GCAUG in a trichromatic splicing reporter assay. In the brain, secondary motifs regulate RNA splicing in neuronal development, enabling a second wave of splicing changes as Rbfox levels increase. Additionally, secondary motifs are activated in neuronal subtypes according to cellular gene expression levels of Rbfox, contributing to tissue diversity in the mammalian brain.

This work presents the first observation of spatiotemporal regulation through suboptimal motifs in RNA-binding proteins, a phenomenon that may be a widespread. Furthermore, the characterization of Rbfox secondary motifs reveals new regulatory targets of an essential splicing factor, which may contribute to mammalian brain development, synaptic plasticity, and human disease.