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Two overlapping reading frames in a single exon encode interacting proteins--a novel way of gene usage.

ABSTRACT: The >1 kb XL-exon of the rat XLalphas/Galphas gene encodes the 37 kDa XL-domain, the N-terminal half of the 78 kDa neuroendocrine-specific G-protein alpha-subunit XLalphas. Here, we describe a novel feature of the XL-exon, the presence of an alternative >1 kb open reading frame (ORF) that completely overlaps with the ORF encoding the XL-domain. The alternative ORF starts 32 nucleotides downstream of the start codon for the XL-domain and is terminated by a stop codon exactly at the end of the XL-exon. The alternative ORF encodes ALEX, a very basic (pI 11.8), proline-rich protein of 356 amino acids. Both XLalphas and ALEX are translated from the same mRNA. Like XLalphas, ALEX is expressed in neuroendocrine cells and tightly associated with the cytoplasmic leaflet of the plasma membrane. Remarkably, ALEX binds to the XL-domain of XLalphas. Our results reveal a mechanism of gene usage that is without precedent in mammalian genomes.

SUBMITTER: Klemke M

PROVIDER: S-EPMC125537 | biostudies-literature | 2001 Jul

REPOSITORIES: biostudies-literature

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Two overlapping reading frames in a single exon encode interacting proteins--a novel way of gene usage.

Klemke M M Kehlenbach R H RH Huttner W B WB

The EMBO journal 20010701 14

The >1 kb XL-exon of the rat XLalphas/Galphas gene encodes the 37 kDa XL-domain, the N-terminal half of the 78 kDa neuroendocrine-specific G-protein alpha-subunit XLalphas. Here, we describe a novel feature of the XL-exon, the presence of an alternative >1 kb open reading frame (ORF) that completely overlaps with the ORF encoding the XL-domain. The alternative ORF starts 32 nucleotides downstream of the start codon for the XL-domain and is terminated by a stop codon exactly at the end of the XL- ...[more]

PMID: 11447126

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Project description: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.

Dataset Information

Two overlapping reading frames in a single exon encode interacting proteins--a novel way of gene usage.

Publications

Two overlapping reading frames in a single exon encode interacting proteins--a novel way of gene usage.

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