Efficient co-transcriptional splicing enforces rapid microexon definition and inclusion by SRRM4

Alternative splicing expands the coding potential of the genome. Typical human exons are 150 nucleotides long, encoding 50 amino acids. Microexons are only 3-27 nucleotides long; yet they are important regulators of cellular processes in neurons, muscle, and pancreas. In neurons, microexon inclusion is aided by binding of the neuronal splicing factor SRRM4 to flanking upstream 3' splice sites (3'SSs). Whether this manner of exon definition can be achieved in the timeframe of co-transcriptional splicing is unknown. Here, we employed nascent RNA sequencing to analyze SRRM4-dependent microexons in neuronal cells and found that co-transcriptional microexon splicing is so efficient, the upstream intron is removed before the downstream intron is completely synthesized. This suggests a mechanism for microexon inclusion, whereby co-transcriptional removal of the upstream intron eliminates competition for the microexon's non-canonical downstream 5'SS. We found that strengthening this 5'SS promoted constitutive microexon inclusion independently of SRRM4, indicating that SRRM4 binding alone is a strong stimulator of microexon definition. Thus, SRRM4's role is to promote rapid splicing of the upstream intron, leaving the microexon's non-canonical 5'SS as the only option for further splicing. These physiologically significant splicing events thereby require co-transcriptionality to yield neuronal mRNA isoforms.