LAUSR

Simple Summary The biological relevance of peptides that originate from non-canonical translational initiation sites have been increasingly recognized over the years. Peptides encoded by open reading frames upstream of canonical protein coding sequences are frequently translated and act as translational regulators, contribute to the immunopeptidome as cellular antigens, and are implicated in various cellular functions through peptide–protein interactions or as part of protein complexes. In this review, we first give an overview of the most relevant technical advances in non-canonical peptide detection. In the second part of the review, we focus on the functional implications of uPeptides and delineate how this largely unexplored compartment of the human peptidome affects tumor biology and may offer new opportunities for targeted and immunological cancer therapy. Abstract Recent technological advances have facilitated the detection of numerous non-canonical human peptides derived from regulatory regions of mRNAs, long non-coding RNAs, and other cryptic transcripts. In this review, we first give an overview of the classification of these novel peptides and summarize recent improvements in their annotation and detection by ribosome profiling, mass spectrometry, and individual experimental analysis. A large fraction of the novel peptides originates from translation at upstream open reading frames (uORFs) that are located within the transcript leader sequence of regular mRNA. In humans, uORF-encoded peptides (uPeptides) have been detected in both healthy and malignantly transformed cells and emerge as important regulators in cellular and immunological pathways. In the second part of the review, we focus on various functional implications of uPeptides. As uPeptides frequently act at the transition of translational regulation and individual peptide function, we describe the mechanistic modes of translational regulation through ribosome stalling, the involvement in cellular programs through protein interaction and complex formation, and their role within the human leukocyte antigen (HLA)-associated immunopeptidome as HLA uLigands. We delineate how malignant transformation may lead to the formation of novel uORFs, uPeptides, or HLA uLigands and explain their potential implication in tumor biology. Ultimately, we speculate on a potential use of uPeptides as peptide drugs and discuss how uPeptides and HLA uLigands may facilitate translational inhibition of oncogenic protein messages and immunotherapeutic approaches in cancer therapy.