Structure and Expression of the Guinea Pig Preproenkephalin Gene: Site-Specific Cleavage in the 3′ Untranslated Region Yields Truncated mRNA Transcripts in Specific Brain Regions

Abstract

We isolated the guinea pig preproenkephalin gene from a genomic library by hybridization to a rat cDNA probe. The entire nucleotide sequence of the gene was determined. Genomic Southern blot hybridization demonstrated that the gene exists in a single copy within the genome. On the basis of RNase protection transcript mapping and homology comparisons with known preproenkephalin sequences from other species and assuming a poly(A) tail length of 100 residues, we predicted an mRNA transcript of approximately 1,400 nucleotides encoded by three exons. Northern (RNA) blot analysis of total RNA from several brain regions showed high levels of preproenkephalin mRNA in the caudate putamen, nucleus accumbens, and hypothalamus, with detectable levels in the amygdala, ventral tegmental area, and central gray and also in the pituitary. Unexpectedly, in several brain regions, the mRNA appeared not only in the 1,400-nucleotide length but also in a shorter length of approximately 1,130 bases. Significant amounts of the shorter mRNA were found in the caudate putamen, nucleus accumbens, and amygdala. The longer, but not the shorter, transcripts from the caudate putamen were found to be polyadenylated, but the difference in size was not due solely to the presence of poly(A) tails. Northern gel analysis of total RNA from the caudate putamen with probes from each exon, together with RNase protection mapping of the 3 end of the mRNA demonstrated that the 1,400-base preproenkephalin mRNA transcripts are cleaved in a site-specific manner in some brain regions, yielding a 1,130-base transcript and a 165-base polyadenylated fragment derived from the terminal end of the 3 untranslated region of the mRNA. This cleavage may serve as a preliminary step in RNA degradation and provide a mechanism for control of preproenkephalin mRNA abundance through selective degradation.The preproenkephalin gene is one of three genes, along with the preprodynorphin and preproopiomelanocortin genes, that encode precursor proteins from which are derived all endogenous opioid peptides. These peptides contain the methionineenkephalin ([Met 5 ]enkephalin) or leucine-enkephalin ([Leu 5 ] enkephalin) sequence motif and exhibit a diversity of functions as neurotransmitters, neuromodulators, and hormones. Preproenkephalin is widely expressed in the brain and peripheral nervous system as well as in the pituitary, male and female reproductive tracts, adrenal medulla, gastrointestinal tract, lymphocytes, and other tissues. The preproenkephalin gene sequences from humans, rats, and golden hamsters have been previously reported (46,52,77), as have the cDNA sequences from humans, rats, mice, cattle, and Xenopus laevis (17,26,37,40,45,76,79). Characteristic features of preproenkephalin from mammalian species in which the gene or mRNA structure has been determined include a signal sequence, six cysteine residues in the amino-terminal region of the propeptide, and seven enkephalin-containing peptide sequences flanked by basic amino acid residues. Proteolytic processing of...

Characterization of Antibacterial COOH-terminal Proenkephalin-A-derived Peptides (PEAP) in Infectious Fluids

“…Sequence comparison of bovine PEA 198 -239 with corresponding fragments from several species. PEA sequences were retrieved from the SwissProt or GenBank data base: bovine (P01211) (7), human (P012100) (3,81), pig (JL0067) (82), rat (P04094) (62,63), mouse (P22005) (83), Mesocricetus auratus (Syrian golden hamster) (MAU09941) (84), guinea pig (P47969) (85), Xenopus laevis (P01012) (86), Mytilus edulis and Theromyzon tessulatum (leech) (87). Leuand Met-enkephalins were underlined.…”

DNA Structure Determines Protein Binding and Transcriptional Efficiency of the Proenkephalin cAMP-responsive Enhancer

“…Yet point mutations in or deletion of CRE-1 alters the transcriptional response to cell surface ligands, to second messengers, and to CRE-binding proteins (3,4,9). Comparison of sequences from several species, including human (10), rat (11), hamster (12), and guinea pig (13), reveals that the 27-base pair region containing the CREs is identical. The arrangement of the CREs with respect to each other as well as the context of each CRE must therefore be critical.…”

Gene expression variance based on random sequencing in rat remnant kidney

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