title: Opioid precursor protein isoform is targeted to the cell nuclei in the human brain.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.affiliation: Department of Pharmaceutical Biosciences, Uppsala University, Uppsala 751 24, Sweden; State Key Lab for Molecular Biology, Bogomoletz Institute of Physiology, Kiev 01024, Ukraine.
authors.forename: Olga
authors.initials: O
authors.lastname: Kononenko
authors.affiliation: Department of Pharmaceutical Biosciences, Uppsala University, Uppsala 751 24, Sweden. Electronic address: Igor.Bazov@farmbio.uu.se.
authors.forename: Igor
authors.initials: I
authors.lastname: Bazov
authors.affiliation: Department of Pharmaceutical Biosciences, Uppsala University, Uppsala 751 24, Sweden.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Watanabe
authors.affiliation: Department of Pharmaceutical Biosciences, Uppsala University, Uppsala 751 24, Sweden; Department of Functional Genomics, Institute Molecular Biology, Kiev 03680, Ukraine.
authors.forename: Ganna
authors.initials: G
authors.lastname: Gerashchenko
authors.affiliation: Department of Medical Cell Biology, Uppsala University, 751 23, Sweden.
authors.forename: Oleg
authors.initials: O
authors.lastname: Dyachok
authors.affiliation: Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen 30001, Netherlands.
authors.forename: Dineke S
authors.initials: DS
authors.lastname: Verbeek
authors.affiliation: Department of Forensic Medicine, Karolinska Institute, Stockholm 171 77, Sweden.
authors.forename: Kanar
authors.initials: K
authors.lastname: Alkass
authors.affiliation: Department of Forensic Medicine, Karolinska Institute, Stockholm 171 77, Sweden.
authors.forename: Henrik
authors.initials: H
authors.lastname: Druid
authors.affiliation: Department of Pharmaceutical Biosciences, Uppsala University, Uppsala 751 24, Sweden.
authors.forename: Malin
authors.initials: M
authors.lastname: Andersson
authors.affiliation: Department of Neuroscience, Science for Life Laboratory, Karolinska Institute, Stockholm 171 77, Sweden.
authors.forename: Jan
authors.initials: J
authors.lastname: Mulder
authors.affiliation: Institute of Molecular Medicine-Neurobiology Research, University of Southern Denmark, Odense 5000, Denmark.
authors.forename: Åsa Fex
authors.initials: ÅF
authors.lastname: Svenningsen
authors.affiliation: Department of Psychiatry and Human Behavior, University of Mississippi Medical Center, Jackson 2500, USA.
authors.forename: Grazyna
authors.initials: G
authors.lastname: Rajkowska
authors.affiliation: Department of Psychiatry and Human Behavior, University of Mississippi Medical Center, Jackson 2500, USA.
authors.forename: Craig A
authors.initials: CA
authors.lastname: Stockmeier
authors.affiliation: State Key Lab for Molecular Biology, Bogomoletz Institute of Physiology, Kiev 01024, Ukraine.
authors.forename: Oleg
authors.initials: O
authors.lastname: Krishtal
authors.affiliation: Department of Pharmaceutical Biosciences, Uppsala University, Uppsala 751 24, Sweden.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Yakovleva
authors.affiliation: Department of Pharmaceutical Biosciences, Uppsala University, Uppsala 751 24, Sweden.
authors.forename: Georgy
authors.initials: G
authors.lastname: Bakalkin
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002421:Caudate Nucleus / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D004399:Dynorphins / Q000378:metabolism; D004745:Enkephalins / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology; D020868:Gene Silencing / Q000502:physiology; D006801:Humans; D008297:Male; D008875:Middle Aged; D018847:Opioid Peptides / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D055815:Young Adult
chemical_list: D000596:Amino Acids; D004745:Enkephalins; D018847:Opioid Peptides; D020033:Protein Isoforms; D011498:Protein Precursors; D012333:RNA, Messenger; D004399:Dynorphins; C033660:preproenkephalin
keywords: Alternative splicing; Human brain; Neuropeptide precursor protein; Nuclear localization; Prodynorphin