Compartmentalization is one of the decisive characteristics of cellular life, enabling the cell to build a complex network of enzymatic reactions and metabolic pathways. Drivers of this cellular management are organelles and the vesicles and tubular structures that connect them, all of which are separated from the cytoplasm by a lipid membrane. Intracellular transport, especially secretion to the extracellular space or to the cell membrane, is a highly regulated and organized process. In this work, we focused on the secretion of big protein cargo like chylomicrons and collagens. We aimed to analyze the role of alternative splicing as a regulation mechanism for the secretion of large cargo. Alternative splicing is a mechanism in gene expression where different combinations of exons are included or excluded from mRNA transcripts, leading to the production of multiple protein isoforms from a single gene. We comprehensively analyzed RNA sequencing data from human tissues as well as B-lymphocytes. We found a previously uncharacterized exon in the gene SEC31A that codes for the outer layer of coat protein complex II (COPII), a complex responsible for the transport from the endoplasmic reticulum (ER) to the Golgi-apparatus. Using a correlation analysis with the inclusion levels of the exon and gene expression throughout the human tissue data, we found leads to the functionality and regulation of this alternatively spliced exon. We could show that the inclusion of the exon enhances the transport of lipids in polar differentiated Caco-2 cells. Mini gene experiments demonstrated that the inclusion is regulated by the splicing factor RBM47. Finally, AlphaFold structure prediction with the resulting alternative protein isoforms revealed a change in interaction with COPII partner SEC23A. We also investigated a previously characterized alternative splice site in SEC31A, which leads to a shortened exon. This shortened exon decreased the secretion efficiency of glycosylphosphatidylinositol (GPI)-anchored and e-cadherin cargo in the RUSH assay but enhanced collagen secretion. In a fluorescent recovery after photobleaching (FRAP) experiment, we observed a reduced mobile fraction of COPII puncta with the shortened exon. Lastly, we analyzed RNA sequencing data of differentiating B-lymphocytes as a model for the regulatory effect of the transcriptome on intracellular trafficking and secretion. B-lymphocytes show rather extensive changes in gene expression during their change from memory cells, to plasmablasts, and plasma cells, but alternative splicing changes are most abundant in memory cells. We found multiple splice events, which are unique to memory cells, particularly in PICALM and KLHL12, which could potentially have a significant impact on the function of the proteins. In summary, we were able to showcase the mechanism by which the structure and function of COPII is altered via alternative splicing of SEC31A, and we demonstrated similar mechanisms in B-lymhocytes. With these results, we depended our knowledge of the role that alternative splicing has on secretory specificity and regulation of large cargo secretion.
