Novel kinase inhibitory micropeptides translated from uORFs

About 40% of human mRNAs contain upstream open reading frames (uORFs) in their 5’-untranslated regions. Some of these uORF sequences, thought to attenuate scanning ribosomes or lead to mRNA degradation, were recently shown to be translated, although the function of most encoded peptides remains unknown. Our recent study was the first to reveal a uORF-encoded peptide exhibiting kinase inhibitory activity (Jayaram DR et al. PNAS (2021)). This uORF (uORF2), upstream of a protein kinase C (PKC) family member (PKCeta) main ORF, encodes for a peptide (uPEP2) containing the typical PKC pseudosubstrate (PS) motif present in all PKCs that auto-inhibits their kinase activity. We show direct binding and selective inhibition of the catalytic activity of novel PKCs by uPEP2 (but not classical or atypical PKCs). Functionally, treatment of breast cancer cells with uPEP2 diminished cell survival and their migration and synergized with chemotherapy by interfering with the response to DNA damage. In a xenograft of MDA-MB-231 triple-negative breast cancer mice models, uPEP2 suppressed tumour progression, invasion, and metastasis. The endogenous deletion of uORF2 by CRISPR/Cas9 in the non-tumorigenic MCF-7 cells showed that it impedes cell proliferation, acting as a growth suppressor. Together, we point to a new layer of protein regulation by a uORF-encoded kinase inhibitor, which could present a forerunner for other uORFs, possessing kinase inhibitory function. As kinase inhibitors, uORF-encoded peptides may play a role in signalling and stress, acting in cis and/or trans, forming diverse regulatory networks, thereby opening new views into eukaryotic protein regulation and cancer biology.