Examining protein interaction differences between sorting nexin 2 (SNX2) isoforms created by alternative splicing of a microexon exon, using AP-MS

ABSTRACT: We examined how alternative RNA splicing affects protein interactions at large scale by creating a protein-protein interaction network using data from interaction databases and then examined how interactions might be affected by splicing. We also examined whether alternative RNA splicing often affected protein localisation within cells, which might come about from altered interactions. To do this, we created a targeted library of protein isoforms generated via alternative splicing events overlapping interfaces that mediate interactions, fused to fluorescent proteins. This library was transfected into HeLa cells and examined via high-content imaging. We found many protein isoforms that differed in the presence/absence of a cassette exon differed in localisation. A notable isoform pair detected in the imaging screen was two isoforms of sorting nexin 2 (SNX2) that differed in the inclusion/exclusion of 4-residues in SNX2’s BAR domain, which caused striking localisation differences. To examine this event further and examine potential connections to altered protein interactomes, we knocked out SNX2 in HeLa cells and rescued the knockout with either of the two SNX2 isoforms (untagged). We then used the rescue cell lines for affinity purification mass spectrometry (AP-MS) , using an antibody against SNX2, which recognize either isoform, for pulldown to identify potential differences in interactors. We didn’t find any clear evidence of different interactors for the two SNX2 isoforms, although noted both isoforms interacted with core SNX-BAR proteins, suggesting both isoforms form functional SNX-BAR complexes. We hypothesize that the lack of identified differential interactors may partly be due to loss of more transient interactors in our AP-MS approach. Accordingly, we found the two isoforms interacted differentially with the insulin receptor (INSR) in a LUMIER assay.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Hela Cell

SUBMITTER: Robert Weatheritt  

LAB HEAD: Robert J. Weatheritt

PROVIDER: PXD074758 | Pride | 2026-03-31

REPOSITORIES: Pride