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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.<\/jats:p>","DOI":"10.1038\/s41594-026-01875-8","type":"journal-article","created":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T21:56:35Z","timestamp":1792014995000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Alternative splicing fine-tunes modular protein interfaces to control protein localization"],"prefix":"10.1038","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-2939-1308","authenticated-orcid":false,"given":"Peter","family":"Kjer-Hansen","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karina","family":"Pazaky","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daisy","family":"Kavanagh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3082-7653","authenticated-orcid":false,"given":"Helen E.","family":"King","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-4006-8514","authenticated-orcid":false,"given":"Savannah","family":"O\u2019Connell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gabriela","family":"Santos-Rodriguez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abigail K.","family":"Grootveld","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Helaine Graziele Santos","family":"Vieira","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1838-4976","authenticated-orcid":false,"given":"Scott","family":"Berry","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Javier","family":"Fernandez-Chamorro","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3716-1783","authenticated-orcid":false,"given":"Robert J.","family":"Weatheritt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,10,14]]},"reference":[{"key":"1875_CR1","doi-asserted-by":"publisher","first-page":"1413","DOI":"10.1038\/ng.259","volume":"40","author":"Q Pan","year":"2008","unstructured":"Pan, Q., Shai, O., Lee, L. 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