Abstract
Thousands of human small and alternative open reading frames (smORFs and alt-ORFs, respectively) have recently been annotated. Many alt-ORFs are co-encoded with canonical proteins in multicistronic configurations, but few of their functions are known. Here, we report the detection of alt-RPL36, a protein co-encoded with human RPL36. Alt-RPL36 partially localizes to the endoplasmic reticulum, where it 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 increases plasma membrane PI(4,5)P2 levels, upregulates PI3K-AKT-mTOR signaling, and increases cell size. Alt-RPL36 contains four phosphoserine residues, point mutations of which abolish interaction with TMEM24 and, consequently, alt-RPL36 effects on PI3K signaling and cell size. These results implicate alt-RPL36 as an upstream regulator of PI3K-AKT-mTOR signaling. More broadly, the RPL36 transcript encodes two sequence-independent polypeptides that co-regulate translation via different molecular mechanisms, expanding our knowledge of multicistronic human gene functions.
Highlights
Thousands of human small and alternative open reading frames have recently been annotated
The first stop codon in-frame with the observed tryptic peptides is downstream of the ribosomal protein L36 (RPL36) stop codon, meaning that alt-RPL36 is longer than RPL36 and completely encompasses its coding sequence
We have identified a previously unannotated protein, alt-RPL36, that is co-encoded with human ribosomal protein L36 in the −1 reading frame of RPL36 transcript variant 2
Summary
Thousands of human small and alternative open reading frames (smORFs and alt-ORFs, respectively) have recently been annotated. ORFs, >100 amino acids, respectively) have recently been revealed via genomic and proteomic technologies in mammalian genomes[1,2] These genes previously escaped annotation not just because of their short length, but because, as a class, they exhibit low homology to proteins of known function, and are enriched for initiation at near-cognate non-AUG start codons (~50%)[3,4]. There is proteomic evidence for hundreds of uncharacterized human smORFs and alt-ORFs that are coencoded with annotated proteins and initiate at non-AUG start codons These findings are, in many cases, supported by ribosome profiling, conservation analyses, and in silico prediction of functional domains and secondary structure[13,14,15]. Notable exceptions include the MIEF1 microprotein, which is encoded in a 5′UTR and regulates translation by mitoribosomes[9], and Aw112010, which initiates at a near-cognate start codon and is required for mucosal immunity[10]
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