Ribosome profiling and mass spectrometry have revealed thousands of small and alternative open reading frames (sm/alt-ORFs) that are translated into polypeptides variously termed as microproteins and alt-proteins in mammalian cells. Some micro-/alt-proteins exhibit stress-, cell-type-, and/or tissue-specific expression; understanding this regulated expression will be critical to elucidating their functions. While differential translation has been inferred by ribosome profiling, quantitative mass spectrometry-based proteomics is needed for direct comparison of microprotein and alt-protein expression between samples and conditions. However, while label-free quantitative proteomics has been applied to detect stress-dependent expression of bacterial microproteins, this approach has not yet been demonstrated for analysis of differential expression of unannotated ORFs in the more complex human proteome. Here, we present global micro-/alt-protein quantitation in two human leukemia cell lines, K562 and MOLT4. We identify 12 unannotated proteins that are differentially expressed in these cell lines. The expression of six micro/alt-proteins from cDNA was validated biochemically, and two were found to localize to the nucleus. Thus, we demonstrate that label-free comparative proteomics enables quantitation of micro-/alt-protein expression between human cell lines. We anticipate that this workflow will enable the discovery of regulated sm/alt-ORF products across many biological conditions in human cells.

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.

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, wereport 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 4,5bisphosphate [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.

The triple burden of malnutrition in many low- and middle-income countries (LMICs) is partly a result of changing food environments and a shift from traditional diets to high-calorie Western-style diets. Exploring the relationship between food sources and food- and nutrition-related outcomes is important to understanding how changes in food environments may affect nutrition in LMICs. This study examined associations of household food source with household food insecurity, individual dietary diversity and individual body mass index in Western Kenya. Interview-administered questionnaire and anthropometric data from 493 adults living in 376 randomly-selected households were collected in 2019. Adjusted regression analyses were used to assess the association of food source with measures of food insecurity, dietary diversity and body mass index. Notably, participants that reported rearing domesticated animals for consumption (‘own livestock’) had lower odds of moderate or severe household food insecurity (odds ratio (OR) = 0.29 (95% CI: 0.09, 0.96)) and those that reported buying food from supermarkets had lower odds of moderate or severe household food insecurity (borderline significant, OR = 0.37 (95% CI: 0.14, 1.00)), increased dietary diversity scores (Poisson coefficient = 0.17 (95% CI: 0.10, 0.24)) and higher odds of achieving minimum dietary diversity (OR = 2.84 (95% CI: 1.79, 4.49)). Our findings provide insight into the relationship between food environments, dietary patterns and nutrition in Kenya, and suggest that interventions that influence household food source may impact the malnutrition burden in this context.