TY - JOUR T1 - Alt-RPL36 downregulates the PI3K-AKT-mTOR signaling pathway by interacting with TMEM24 JF - bioRxiv DO - 10.1101/2020.03.04.977314 SP - 2020.03.04.977314 AU - Xiongwen Cao AU - Alexandra Khitun AU - Zhenkun Na AU - Thitima Phoodokmai AU - Khomkrit Sappakhaw AU - Elizabeth Olatunji AU - Chayasith Uttamapinant AU - Sarah A. Slavoff Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/03/05/2020.03.04.977314.abstract N2 - While thousands of previously unannotated small and alternative open reading frames (alt-ORFs) have recently been revealed in the human genome, the functions of only a handful are currently known, and no post-translational modifications of their polypeptide products have yet been reported, leaving open the question of their biological significance as a class. Using a proteomic strategy for discovery of unannotated short open reading frames in human cells, we report the detection of alt-RPL36, a 148-amino acid protein co-encoded with and overlapping human RPL36. Alt-RPL36 interacts with TMEM24, which transports the phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] precursor phosphatidylinositol from the endoplasmic reticulum to the plasma membrane. Knock-out of alt-RPL36 in HEK 293T cells increased PI(4,5)P2 levels in the plasma membrane and upregulated the PI3K-AKT-mTOR signaling pathway. Remarkably, we find that four serine residues of alt-RPL36 are phosphorylated, and mutation of these four serines to alanine abolished the interaction with TMEM24 and regulation of PI3K signaling. These results implicate alt-RPL36 as a novel regulator of PI(4,5)P2 synthesis upstream of the PI3K-AKT-mTOR signaling pathway, and the first example of a phosphorylated alt-ORF product. More broadly, both alt-RPL36 and RPL36 regulate protein synthesis and cell growth via different molecular mechanisms – PI3K signaling and ribosome composition, respectively. One human transcript can therefore express two sequence-independent polypeptides from overlapping ORFs that regulate the same processes via distinct mechanisms. ER -