Pooled overexpression screening identifies PIPPI as a novel microprotein involved in the ER stress response

Microproteins encoded by short open reading frames (sORFs) of <100 codons have been predicted to constitute a substantial fraction of the eukaryotic proteome. However, the relevance and roles of a majority of microproteins remain undefined, as only a small fraction of these intriguing cellular players have been characterized in-depth so far. Here, we use pooled overexpression screens with a library of 11 338 sORFs to overcome the challenge of elucidating which of the thousands of putative translated sORFs are biologically functional. As a proof-of-concept, we performed a phenotypic screen to identify sORFs protecting cells from treatment with the nucleotide analogue 6-thioguanine. With this approach, we identified two cytoprotective microproteins: altDDIT3 and PIPPI. PIPPI is encoded within the LCR16a core duplicon of the Morpheus/NPIP gene cluster. We show that PIPPI modulates the cellular response to protein folding stress in the endoplasmic reticulum (ER) and interacts with proteins in the same pathway, including protein disulfide isomerase ERp44. PIPPI overexpression protects, while PIPPI knockdown sensitizes cells to ER stress. Besides providing mechanistic insights into a new microprotein, this study highlights the power of using pooled overexpression screens to identify functional microproteins.