The Emerging Role of uORF-Encoded uPeptides and HLA uLigands in Cellular and Tumor Biology

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.

Keywords: HLA uLigands; cancer; immunotherapy; non-canonical peptides; translation; uORFs; uPeptides.

Figure 1

Intra- and extracellular functions of human uORF-encoded peptides. The nascent peptide (I) can induce ribosome stalling, mostly followed by transcript degradation, resulting in reduced translation of the main protein CDS, indicated by the crossed out arrow. After proteasomal degradation and processing via the MHC-related antigen presenting machinery, uPeptides contribute to the immunopeptidome (II). The uPeptides contribute to the micropeptidome (III) and may affect diverse cellular functions through interaction with key regulatory proteins or as part of protein complexes.

Figure 2

Mechanisms of ribosome stalling-mediated translational regulation. (A) Under physiological conditions, ribosomes may scan through the uORF start codon (leaky scanning) or reinitiate at the CDS after translating the uORF, maintaining normal CDS translation and cellular homeostasis. (B) Interaction of the nascent peptide with small molecules, metabolites or other molecular interactors may induce ribosome stalling, preventing ribosomal reinitiation, and leading to transcript degradation via nonsense-mediated decay (NMD). (C) Mutation-associated introduction of a new uStop codon within the uORF sequence may lead to ribosome stalling and can prohibit downstream translation, indicated by the crossed out arrow.

Figure 3

Non-canonical uPeptide-derived neoantigens. (A) A large fraction of cellular proteins undergo proteasomal degradation upon ubiquitylation (+Ub) and will partly be presented on the cell surface as HLA ligands. (B) Upon tumorigenesis, diverse mechanisms including the activation of the ISR or alternative splicing may enable translation of previously skipped or non-existing uORFs (orange box). Somatic mutations in de novo uORF start (uStart) sites or internal uORF sequence may result in novel uPeptides or changes in uPeptide sequences, altering the micropeptidome compared to healthy cells. Proteasomal degradation of the novel uPeptides generates cancer cell-specific HLA complexes, which may be targetable by immunotherapeutic antigens or cytotoxic T-cells, indicated by inhibitory arrows.

Figure 4

Effects of PKC-η uAUG.2 on protein kinase activity and downstream function [125]. (A) The uAUG.2 peptide (orange square) encoded from the PKC-η TLS inhibits the catalytic activity of novel PKCs (blue circles). (B) Increased uPeptide levels can lead to reduced PKC activity, resulting in decreased (↓) cell proliferation, invasion and metastasis and increased (↑) cell death. Overexpression of uAUG.2 (I) or direct application of the AUG.2 uPeptide as a small peptide drug (II) may offer novel ways of protein kinase-inhibition in cancer therapy.