Savannah O’COnnell

Alternative splicing provides a pervasive means to expand proteome complexity, yet how it reorganizes protein interactions and cellular localization remains unclear. Here, we show that tissue-regulated longer exons preferably reshape local interaction patterns, whereas microexons fine-tune key interfaces linking distinct cellular processes. Integration of subcellular localization data further indicates that such rewiring can redistribute proteins within cells. By using a high-content imaging approach to systematically evaluate the influence of individual exons on protein localization, we show that 38% of the tested isoform pairs alter localization, with microexons, albeit typically shorter than five amino acids, accounting for a substantial proportion of these effects. Bioinformatic and structural analyses demonstrate that microexons can extend secondary structural regions and reposition charged residues, suggesting a potential to modulate local electrostatic environments. Consistent with this, biochemical analysis of a 4-aa microexon in sorting nexin 2, identified in our screen, confirms that residue insertion rather than side-chain chemistry drives key differences in protein localization through repositioning of a flanking, charged residue. Together, these findings suggest that tissue-regulated alternative splicing fine-tunes interface architecture, thus regulating protein assembly, localization and proteome organization.

Alternative splicing provides a pervasive means to expand proteome complexity, yet how it reorganises protein interactions and constrains where proteins act within cell remains unclear. By constructing an interface-resolved interaction network composed of 17,660 experimentally defined contact sites, we reveal that tissue-specific alternative splicing remodels protein connectivity by reshaping modular protein architecture. Longer exons reshape local interaction patterns whereas microexons fine-tune key interfaces linking distinct cellular processes. Integration of subcellular localisation data further indicates that such rewiring can redistribute proteins within cells. To test this, we developed a high-content imaging approach to systematically evaluate the influence of individual exons on protein localisation and screened a targeted library of protein isoforms differing in individual exons. 38% of the tested isoform pairs altered localisation, with microexons, although typically shorter than five amino acids, accounting for a substantial proportion of these effects. Bioinformatic and structural analyses identified that microexons can extend secondary structural regions and reposition charged residues, suggesting a potential to modulate local electrostatic environments. Consistent with this, biochemical analysis of a four–amino acid microexon in sorting nexin 2 - identified through our screen - confirmed that residue insertion, rather than side chain chemistry, was driving differences in protein localisation through repositioning of a flanking, charged residue. Together, these findings describe a principle by which alternative splicing fine-tunes interface architecture to coordinate protein assembly, localisation, and proteome organisation.

Pancreatic cancer (PC) is a highly metastatic malignancy. More than 80% of patients with PC present with advanced-stage disease, preventing potentially curative surgery. The neuropeptide Y (NPY) system, best known for its role in controlling energy homeostasis, has also been shown to promote tumorigenesis in a range of cancer types, but its role in PC has yet to be explored. We show that expression of NPY and NPY1R are up-regulated in mouse PC models and human patients with PC. Moreover, using the genetically engineered, autochthonous KP ^R172H C mouse model of PC, we demonstrate that pancreas-specific and whole-body knockout of Npy1r significantly decreases metastasis to the liver. We identify that treatment with the NPY1R antagonist BIBO3304 significantly reduces KP ^R172H C migratory capacity on cell-derived matrices. Pharmacological NPY1R inhibition in an intrasplenic model of PC metastasis recapitulated the results of our genetic studies, with BIBO3304 significantly decreasing liver metastasis. Together, our results reveal that NPY/NPY1R signaling is a previously unidentified antimetastatic target in PC.