Core splicing architecture and early spliceosomal recognition determine microexon sensitivity to SRRM3/4

Microexons are essential for proper functioning of neurons and pancreatic endocrine cells, where their inclusion depends on the splicing factors SRRM3/4. However, in pancreatic cells, their lower expression limits inclusion to only the most sensitive neuronal microexons. Although various cis-acting elements are known to contribute to microexon regulation, how they determine this differential dose response and sensitivity to SRRM3/4 remains unknown. Here, through Massively Parallel Splicing Assays probing 28,535 variants, we found that sensitivity to SRRM4 is conserved across vertebrates and overall consistent with a regulatory model whereby the main determinants of microexon sensitivity are related to differences in the interplay between core splicing architecture and length constraints. This conclusion is further supported by distinct spliceosome activity in the absence of SRRM3/4 and by a mathematical model that assumes that the two types of microexons differ only in their efficiency of recruitment of early spliceosomal components.