Olga Kononenko1, Igor Bazov2, Hiroyuki Watanabe3, Ganna Gerashchenko4, Oleg Dyachok5, Dineke S Verbeek6, Kanar Alkass7, Henrik Druid7, Malin Andersson3, Jan Mulder8, Åsa Fex Svenningsen9, Grazyna Rajkowska10, Craig A Stockmeier10, Oleg Krishtal11, Tatiana Yakovleva3, Georgy Bakalkin3. 1. Department of Pharmaceutical Biosciences, Uppsala University, Uppsala 751 24, Sweden; State Key Lab for Molecular Biology, Bogomoletz Institute of Physiology, Kiev 01024, Ukraine. 2. Department of Pharmaceutical Biosciences, Uppsala University, Uppsala 751 24, Sweden. Electronic address: Igor.Bazov@farmbio.uu.se. 3. Department of Pharmaceutical Biosciences, Uppsala University, Uppsala 751 24, Sweden. 4. Department of Pharmaceutical Biosciences, Uppsala University, Uppsala 751 24, Sweden; Department of Functional Genomics, Institute Molecular Biology, Kiev 03680, Ukraine. 5. Department of Medical Cell Biology, Uppsala University, 751 23, Sweden. 6. Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen 30001, Netherlands. 7. Department of Forensic Medicine, Karolinska Institute, Stockholm 171 77, Sweden. 8. Department of Neuroscience, Science for Life Laboratory, Karolinska Institute, Stockholm 171 77, Sweden. 9. Institute of Molecular Medicine-Neurobiology Research, University of Southern Denmark, Odense 5000, Denmark. 10. Department of Psychiatry and Human Behavior, University of Mississippi Medical Center, Jackson 2500, USA. 11. State Key Lab for Molecular Biology, Bogomoletz Institute of Physiology, Kiev 01024, Ukraine.
Abstract
BACKGROUND: Neuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the κ-opioid receptor. Alternative mRNA splicing of neuropeptide genes may regulate cell- and tissue-specific neuropeptide expression and produce novel protein isoforms. We here searched for novel PDYN mRNA and their protein product in the human brain. METHODS: Novel PDYN transcripts were identified using nested PCR amplification of oligo(dT) selected full-length capped mRNA. Gene expression was analyzed by qRT-PCR, PDYN protein by western blotting and confocal imaging, dynorphin peptides by radioimmunoassay. Neuronal nuclei were isolated using fluorescence-activated nuclei sorting (FANS) from postmortem human striatal tissue. Immunofluorescence staining and confocal microscopy was performed for human caudate nucleus. RESULTS: Two novel human PDYN mRNA splicing variants were identified. Expression of one of them was confined to the striatum where its levels constituted up to 30% of total PDYN mRNA. This transcript may be translated into ∆SP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). ∆SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted to the cell nuclei in a model cellular system. The endogenous PDYN protein was identified in the cell nuclei in human striatum by western blotting of isolated neuronal nuclei, and by confocal imaging. CONCLUSIONS AND GENERAL SIGNIFICANCE: High levels of alternatively spliced ∆SP-PDYN mRNA and nuclear localization of PDYN protein suggests a nuclear function for this isoform of the opioid peptide precursor in human striatum.
BACKGROUND: Neuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the κ-opioid receptor. Alternative mRNA splicing of neuropeptide genes may regulate cell- and tissue-specific neuropeptide expression and produce novel protein isoforms. We here searched for novel PDYN mRNA and their protein product in the human brain. METHODS: Novel PDYN transcripts were identified using nested PCR amplification of oligo(dT) selected full-length capped mRNA. Gene expression was analyzed by qRT-PCR, PDYN protein by western blotting and confocal imaging, dynorphin peptides by radioimmunoassay. Neuronal nuclei were isolated using fluorescence-activated nuclei sorting (FANS) from postmortem human striatal tissue. Immunofluorescence staining and confocal microscopy was performed for human caudate nucleus. RESULTS: Two novel humanPDYN mRNA splicing variants were identified. Expression of one of them was confined to the striatum where its levels constituted up to 30% of totalPDYN mRNA. This transcript may be translated into ∆SP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). ∆SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted to the cell nuclei in a model cellular system. The endogenous PDYN protein was identified in the cell nuclei in human striatum by western blotting of isolated neuronal nuclei, and by confocal imaging. CONCLUSIONS AND GENERAL SIGNIFICANCE: High levels of alternatively spliced ∆SP-PDYN mRNA and nuclear localization of PDYN protein suggests a nuclear function for this isoform of the opioid peptide precursor in human striatum.
Authors: Xuan V Nguyen; James Masse; Ashok Kumar; Rattanavijit Vijitruth; Cynthia Kulik; Mei Liu; Dong-Young Choi; Thomas C Foster; Ivan Usynin; Georgy Bakalkin; Guoying Bing Journal: Behav Brain Res Date: 2005-03-24 Impact factor: 3.332