Alternative exon splicing reveals hidden mitochondrial targeting of PLIN3

Abstract Perilipin 3 (PLIN3) is a lipid droplet–associated protein implicated in cellular lipid metabolism. We characterize a previously unrecognized alternative splicing event in human PLIN3 . The generated transcript PLIN3B is detectable at the RNA level across human tissues and is modulated by antisense oligonucleotide–mediated splicing. Despite clear evidence for PLIN3B transcript expression, endogenous PLIN3B protein could not be detected using multiple complementary state-of-the-art detection strategies. When expressed exogenously, PLIN3B coding sequence encodes an unstable protein that localizes predominantly within mitochondria, in contrast to canonical PLIN3. Using super-resolution microscopy, correlative light–electron microscopy, and interaction proteomics, we show that PLIN3B accumulates intramitochondrially and engages protein quality control machinery. Mitochondrial targeting depends on residues shared by both isoforms, not altered by alternative splicing, indicating that mitochondrial targeting is normally repressed. Together, these results show that alternative exon splicing in PLIN3 reveals latent mitochondrial targeting without producing detectable endogenous protein, underscoring the need to distinguish transcript diversity from stable protein expression.