Opioid precursor protein isoform is targeted to the cell nuclei in the human brain

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 New PDYN transcripts were identified using nested PCR amplified from 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 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 ΔSP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). ΔSP-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 ΔSP-PDYN mRNA and nuclear localization of PDYN protein suggest an essential nuclear function for this isoform of the opioid peptide precursor in human striatum.

Epigenetic and Transcriptional Control of the Opioid Prodynorphine Gene: In-Depth Analysis in the Human Brain

“…∆SP -PDYN mRNA contains two introns and three exons, and produces the ∆SP-PDYN protein that is translated from the Met 14 and therefore lacks thirteen amino acids of the signal peptide ( Figure 2 b and Figure 3 a). The ∆SP/NLS-PDYN protein in addition lacks α-neoendorphin and dynorphin A sequences [ 46 ]. ∆SP -PDYN mRNA is expressed in the striatum where its levels constitute approximately 30% of total PDYN mRNA, while in other brain areas its expression is negligible.…”

“…∆SP -PDYN mRNA is expressed in the striatum where its levels constitute approximately 30% of total PDYN mRNA, while in other brain areas its expression is negligible. ∆SP-PDYN protein has a putative bipartite nuclear localization signal (NLS) that has a high score and is located in the opioid domain [ 46 ]. The NLS targets ∆SP-PDYN protein to the cell nuclei.…”

“…The NLS targets ∆SP-PDYN protein to the cell nuclei. Biochemical methods and confocal imaging identified endogenous PDYN protein in the nucleus of neurons in the human striatum ( Figure 3 b–d) [ 46 ]. Consistently, electron microscopic analysis of rat nucleus accumbens demonstrated the presence of Pdyn protein and dynorphin A in the neuronal nuclei along with its location in the smooth endoplasmic reticulum [ 47 ].…”

“…T1 and T2 are transcribed from the sites located between sequences coding for α-neoendorphin and dynorphin A. TSSs in exons are not unique for PDYN and were identified in other genes (Figure 2b) [45]. [46]. Exon 4 of human PDYN contains neuropeptide-encoding sequences, and also exhibits a promoter activity enabling transcription of T1-and T2-PDYN mRNAs from the intragenic TSSs.…”

Mechanical property characterization of hundreds of single nuclei based on microfluidic constriction channel

“…The approaches for the isolation of nuclei can be generally categorized into mechanical‐, and chemical‐based methods . For the former, micropipette , and Dounce homogenizer are commonly utilized to disrupt the structure of cell membrane using the shear force generated during operations. The nuclei are then harvested in the subsequent separation and isolation processes.…”