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Upstream Open Reading Frames Located in the Leader of Protein Kinase M? mRNA Regulate Its Translation.

ABSTRACT: For protein synthesis that occurs locally in dendrites, the translational control mechanisms are much more important for neuronal functioning than the transcription levels. Here, we show that uORFs (upstream open reading frames) in the 5' untranslated region (5'UTR) play a critical role in regulation of the translation of protein kinase M? (PKM?). Elimination of these uORFs activates translation of the reporter protein in vitro and in primary cultures of rat hippocampal neurons. Using cell-free translation systems, we demonstrate that translational initiation complexes are formed only on uORFs. Further, we address the mechanism of translational repression of PKM? translation, by uORFs. We observed an increase in translation of the reporter protein under the control of PKM? leader in neuronal culture during non-specific activation by picrotoxin. We also show that such a mechanism is similar to the mechanism seen in cell stress, as application of sodium arsenite to neuron cultures induced translation of mRNA carrying PKM? 5'UTR similarly to picrotoxin activation. Therefore, we suppose that phosphorylation of eIF2a, like in cell stress, is a main regulator of PKM? translation. Altogether, our findings considerably extend our understanding of the role of uORF in regulation of PKM? translation in activated neurons, important at early stages of LTP.

SUBMITTER: Bal NV

PROVIDER: S-EPMC5061749 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Upstream Open Reading Frames Located in the Leader of Protein Kinase Mζ mRNA Regulate Its Translation.

Bal Natalia V NV Susorov Denis D Chesnokova Ekaterina E Kasianov Artem A Mikhailova Tatiana T Alkalaeva Elena E Balaban Pavel M PM Kolosov Peter P

Frontiers in molecular neuroscience 20161013

For protein synthesis that occurs locally in dendrites, the translational control mechanisms are much more important for neuronal functioning than the transcription levels. Here, we show that uORFs (upstream open reading frames) in the 5' untranslated region (5'UTR) play a critical role in regulation of the translation of protein kinase Mζ (PKMζ). Elimination of these uORFs activates translation of the reporter protein <i>in vitro</i> and in primary cultures of rat hippocampal neurons. Using cel ...[more]

PMID: 27790092

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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.

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Upstream Open Reading Frames Located in the Leader of Protein Kinase M? mRNA Regulate Its Translation.

Publications

Upstream Open Reading Frames Located in the Leader of Protein Kinase Mζ mRNA Regulate Its Translation.

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