Role Of uORFs Research Articles

Upstream open reading frames regulate translation of cancer-associated transcripts and encode HLA-presented immunogenic tumor antigens

BackgroundUpstream open reading frames (uORFs) represent translational control elements within eukaryotic transcript leader sequences. Recent data showed that uORFs can encode for biologically active proteins and human leukocyte antigen (HLA)-presented peptides in malignant and benign cells suggesting their potential role in cancer cell development and survival. However, the role of uORFs in translational regulation of cancer-associated transcripts as well as in cancer immune surveillance is still incompletely understood.MethodsWe examined the translational regulatory effect of 29 uORFs in 13 cancer-associated genes by dual-luciferase assays. Cellular expression and localization of uORF-encoded peptides (uPeptides) were investigated by immunoblotting and immunofluorescence-based microscopy. Furthermore, we utilized mass spectrometry-based immunopeptidome analyses in an extensive dataset of primary malignant and benign tissue samples for the identification of naturally presented uORF-derived HLA-presented peptides screening for more than 2000 uORFs.ResultsWe provide experimental evidence for similarly effective translational regulation of cancer-associated transcripts through uORFs initiated by either canonical AUG codons or by alternative translation initiation sites (aTISs). We further demonstrate frequent cellular expression and reveal occasional specific cellular localization of uORF-derived peptides, suggesting uPeptide-specific biological implications. Immunopeptidome analyses delineated a set of 125 naturally presented uORF-derived HLA-presented peptides. Comparative immunopeptidome profiling of malignant and benign tissue-derived immunopeptidomes identified several tumor-associated uORF-derived HLA ligands capable to induce multifunctional T cell responses.ConclusionOur data provide direct evidence for the frequent expression of uPeptides in benign and malignant human tissues, suggesting a potentially widespread function of uPeptides in cancer biology. These findings may inspire novel approaches in direct molecular as well as immunotherapeutic targeting of cancer-associated uORFs and uPeptides.

A comprehensive catalog of predicted functional upstream open reading frames in humans

Upstream open reading frames (uORFs) latent in mRNA transcripts are thought to modify translation of coding sequences by altering ribosome activity. Not all uORFs are thought to be active in such a process. To estimate the impact of uORFs on the regulation of translation in humans, we first circumscribed the universe of all possible uORFs based on coding gene sequence motifs and identified 1.3 million unique uORFs. To determine which of these are likely to be biologically relevant, we built a simple Bayesian classifier using 89 attributes of uORFs labeled as active in ribosome profiling experiments. This allowed us to extrapolate to a comprehensive catalog of likely functional uORFs. We validated our predictions using in vivo protein levels and ribosome occupancy from 46 individuals. This is a substantially larger catalog of functional uORFs than has previously been reported. Our ranked list of likely active uORFs allows researchers to test their hypotheses regarding the role of uORFs in health and disease. We demonstrate several examples of biological interest through the application of our catalog to somatic mutations in cancer and disease-associated germline variants in humans.