Alternative splicing is a major mechanism for expanding the transcriptomic and proteomic diversity of mammalian cells. Among these events, the inclusion of highly conserved microexons (3-27 nucleotides) is specifically enriched in neurons, where they contribute to cell-specific function. Recent evidence suggests that a subset of neuronal microexons is also expressed in pancreatic beta-cells, reflecting that microexon regulation may be more broadly utilised, although this remains unclear.
By analysing cell-type-specific bulk RNA-seq data from mouse tissue, we identified the selective inclusion of a subset of neuronal microexons in diverse endocrine cell types, including pancreatic beta-cells, adrenal medulla, pituitary gland, and enteroendocrine cells of the gut, which occurs independently of gene expression changes. Furthermore, splicing factors SRRM3 and SRRM4 were likely regulators of the shared neural-endocrine microexons, as SRRM3/4 knockdown in N2A neural cells reduced their inclusion, whereas SRRM3/4 overexpression in HEK293 cells promoted their inclusion in an otherwise non-neuronal context. Moreover, SRRM3/4 expression correlated highly with the inclusion of endocrine microexons, which wasn’t observed for other microexon-regulating splicing factors.
To extend these findings to endocrine cell types not captured by bulk RNA-seq, we examined single-cell RNA-seq data from tissues containing these cell types. SRRM3/4 were expressed in endocrine populations across multiple tissues, including pancreas, intestine, adrenal medulla, pituitary gland, pineal gland, and rare populations in the thymus, lung and prostate, suggesting a conserved regulatory role across endocrine cells that originated from different germ layers. Permutation testing confirmed significant co-expression of SRRM3/4 with canonical endocrine cell markers. Using pseudotime and RNA velocity analyses, we demonstrate that SRRM3/4 is absent in stem and progenitor populations, but is upregulated alongside endocrine markers throughout differentiation, both during development and in replenishing adult tissue. Collectively, our findings identify a conserved program of microexon-mediated splicing regulation across endocrine cells regulated by RNA-binding proteins SRRM3 and SRRM4.