PXD074758

PXD074758 is an original dataset announced via ProteomeXchange.

Title	Examining protein interaction differences between sorting nexin 2 (SNX2) isoforms created by alternative splicing of a microexon exon, using AP-MS
Description	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.
HostingRepository	PRIDE
AnnounceDate	2026-03-31
AnnouncementXML	Submission_2026-03-31_03:15:46.344.xml
DigitalObjectIdentifier	https://doi.org/10.6019/PXD074758
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Robert Weatheritt
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2026-02-23 23:25:13	ID requested
⏵ 1	2026-03-31 03:15:46	announced

10.1101/2025.09.30.679652;

10.6019/PXD074758;

submitter keyword: Alternative splicing, AP-MS, isoforms, microexon,LC-MSMS

Robert J. Weatheritt
contact affiliation	EMBL Australia, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australia
contact email	r.weatheritt@garvan.org.au
lab head
Robert Weatheritt
contact affiliation	UNSW Sydney
contact email	r.weatheritt@garvan.org.au
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD074758
     1. Label: PRIDE project
     2. Name: Examining protein interaction differences between sorting nexin 2 (SNX2) isoforms created by alternative splicing of a microexon exon, using AP-MS