Conservation of extended sequence and structure in the branchpoint-to-3’ splice site region upstream of neural microexons

Abstract Microexons are short exons that are highly conserved in vertebrates and are essential for neurodevelopment. Their small size poses a challenge for regulatory protein binding and exon-definition splice site recognition, which typically relies on standard length exons. Here, we determine the sequence and RNA structural features of neural microexons in humans and in the chick developmental model organism. We demonstrate that a subset of neural microexons undergoes dynamic, stage-specific regulation during chick embryonic brain development that correlates with expression of known microexon regulators, SRRM4 and NOVA1 . Using experimental RNA structure-probing on a subset of neural microexons, we show that shared RNA secondary structures between orthologous human and chicken microexon precursor mRNAs primarily occur in regions of high sequence conservation. We find that both human and chicken neural microexons have extended functional distance between the branchpoint and the 3’ splice. Structurally, branchpoint-to-splice site regions are unusually accessible and relatively unpaired compared to other exon classes. Our data suggest that microexon splicing relies on structural accessibility of the branch-point-to-splice site region, which may influence accessibility for SRRM4 binding and alleviate steric constraints for spliceosome assembly. Graphical Abstract