Translational control of germ cell-expressed mRNA imposed by alternative splicing: opioid peptide gene expression in rat testis.

The three genes encoding the opioid peptide precursors (prodynorphin, proenkephalin, and proopiomelanocortin) are expressed in the rat testis. The sizes of the three opioid mRNAs in the testis differ from the sizes of the corresponding mRNAs in other rat tissues in which these genes are expressed. The smaller testicular proopiomelanocortin mRNA has previously been demonstrated to arise from alternative transcriptional initiation. In the present study, we found that the smaller testicular prodynorphin mRNA, expressed in Sertoli cells, results from alternative mRNA processing. Exon 2, which makes up 5' untranslated sequence, is removed from the mature transcript. Polysome analysis of brain and testis RNA indicates that the alteration of the prodynorphin leader sequence in the testis-specific transcript does not affect the efficiency of translation of this mRNA. The larger testicular proenkephalin transcript, expressed in developing germ cells, also results from alternative mRNA processing. Alternative acceptor site usage in the splicing of intron A results in a germ cell-specific proenkephalin transcript with a 491-nucleotide 5' untranslated leader sequence preceding the preproenkephalin-coding sequence. Polysome analysis indicates that this germ cell-specific proenkephalin mRNA is not efficiently translated. Mechanisms by which alternative mRNA splicing may serve to confer translational regulation upon the testicular proenkephalin transcript are discussed.

Human chorionic gonadotropin regulates expression of the proenkephalin gene in adult rat Leydig cells.

The gene encoding the proenkephalin precursor is expressed in both progenitor spermatogenic cells and somatic cells in the rat testis. The 1750-nucleotide (nt) germ cell-specific proenkephalin mRNA is the predominant form detected in the adult rat. The 1400-nt somatic cell form has previously been shown to be expressed in cultured Sertoli cells derived from sexually immature rats, and FSH treatment increases expression of the proenkephalin gene in that experimental system. In the present report we demonstrate that the 1400-nt proenkephalin transcript is present in a freshly isolated, enriched preparation of adult rat Leydig cells. This gene continues to be expressed when the Leydig cells are maintained in primary culture. Treatment of cultured Leydig cells with hCG or a cAMP analog leads to a rapid increase in proenkephalin-mRNA levels. The expression of the proenkephalin gene throughout rat development in the two major somatic cell types of the testis further suggests that proenkephalin-derived peptides play an important role in modulating testicular function. The gene encoding another of the opioid peptide precursors, POMC, has previously been reported to be expressed in rat Leydig cells. We were unable to detect POMC transcripts in cultured Leydig cells. POMC mRNA is abundant in a germ cell-enriched cell fraction.

Identification of a cyclic adenosine monophosphate-responsive element in the rat corticotropin-releasing hormone gene.

The molecular mechanisms involved in the regulation of expression of the rat CRH gene have been examined in rat pheochromocytoma (PC-12) cells transiently transfected with a chimeric gene containing 1.4 kilobases of rat CRH 5'-flanking DNA fused to the bacterial reporter gene encoding chloramphenicol acetyltransferase. Cyclic AMP analogs and activators of adenylate cyclase positively regulate the expression of this chimeric gene in PC-12 cells, inducing chloramphenicol acetyltransferase activity more than 15-fold. The DNA sequence required for this response to cAMP has been localized to a 59 base pair region located between 238 and 180 base pairs 5' to the putative CRH mRNA cap site. This sequence can confer cAMP-responsiveness on a heterologous promoter in an orientation independent fashion and has homology to cAMP regulatory regions from a number of other eukaryotic genes.

Sertoli cells are the primary site of prodynorphin gene expression in rat testis: regulation of mRNA and secreted peptide levels by cyclic adenosine 3' ,5'-monophosphate analogs in cultured cells.

Prodynorphin is one of three endogenous opioid peptide genes expressed in testis. Through the use of cell fractionation procedures and Northern blot analysis, Sertoli cells were found to be the primary site of prodynorphin mRNA synthesis in rat testis. In situ hybridization of a prodynorphin cRNA probe to fixed adult tissue confirmed this result. Treatment of primary cultures of rat Sertoli cells with a cAMP analog, 8-(4-chlorophenylthio)cAMP, resulted in a transient 5.6-fold increase in steady state prodynorphin mRNA levels relative to those in control cells. This increase was maximal at 48 h of treatment, after which mRNA levels gradually declined. Treatment of Sertoli cells with cAMP analogs resulted in concurrent 2.6-fold decreases in sulfated glycoprotein-2 mRNA levels. Culture medium from Sertoli cells showed a 3.1-fold increase in secreted dynorphin immunoreactivity after treatment with 8-(4-chlorophenylthio)cAMP. Chromatographic analysis indicates that the majority of the immunoreactive dynorphin peptide synthesized in Sertoli cells is present as high mol wt species, with some processing to bioactive peptides.

Regulated expression of the prodynorphin gene in the R2C Leydig tumor cell line.

We report here that prodynorphin mRNA and prodynorphin-derived peptides are synthesized in the R2C rat Leydig tumor cell line. The size of the prodynorphin transcript found in these cells (approximately 2200 nucleotides) is identical to that found in the intact testis. R2C cells also contain proteolytically processed prodynorphin-derived peptides. In R2C cells, the endogenous prodynorphin gene and cellular levels of prodynophin-derived peptides are positively regulated by cAMP analogs, while phorbol esters exert a slight negative regulation of the prodynorphin mRNA. Using gene transfer techniques, we have identified a 210-basepair fragment of the rat prodynorphin gene which initiates the transcription of the bacterial reporter molecule, chloramphenicol acetyl transferase. The chimeric fusion gene, when transfected into R2C cells, exhibited the same positive response to cAMP analogs as the endogenous gene. The results suggest that a cAMP regulatory element resides within the cloned rat prodynorphin fragment, and that the element is functionally active in R2C cells.

The rat corticotropin-releasing hormone gene.

In this paper we have described the isolation and characterization of the rat corticotropin releasing hormone gene. Nucleotide sequence comparisons with the human CRH gene have demonstrated several interesting regions of homology and suggest that the gene was highly conserved through evolution. Additionally we have demonstrated the tissue-specific expression of the rat CRH gene. The regional distribution of expression parallels previously documented immunocytochemical demonstrations and supports the hypothesis that CRH peptides have multiple roles in different tissues. In the peripheral tissues that express CRH mRNA it will be very interesting to document the specific cell type of synthesis by using combined immunocytochemical and in situ histochemical techniques. Additionally we have described initial studies using gene transfer techniques to examine the cAMP responsiveness of the rat CRH gene. We are presently constructing other fusion genes (CRHCAT plasmids) in order to more carefully localize the DNA sequence in the rat CRH gene which mediates this effect, and compare it to the previously reported cAMP-responsive "consensus sequence." Similarly, we also plan to utilize the CRHCAT constructs to examine regulation of the rat CRH gene by glucocorticoids and several other hormone-mediated regulatory pathways. Through these CAT fusion studies we hope to gain a better understanding of the role of certain conserved sequences in the 5' flanking DNA for transcriptional control of the rat (and human) CRH genes.

Structure and expression of the human motilin gene.

The human motilin gene was isolated from a human genomic library and its structure was determined by restriction mapping and DNA sequence analysis. The gene consists of five exons separated by four introns spanning approximately 9 kb of genomic DNA. Exon I encodes the 5' untranslated portion of the motilin mRNA. Exons II and III encode the signal peptide and the 22-amino-acid motilin peptide; codons encoding the motilin moiety are split by an intron. The carboxy-terminal motilin-associated peptide (MAP) is largely encoded by Exons III and IV with the last two amino acids of the motilin precursor and the 3' untranslated region encoded by Exon V. Thus, the motilin gene has an unusual structure in which a small bioactive peptide is encoded on two distinct exons. Examination of the expression of the human and nonhuman primate motilin gene by Northern hybridization analysis indicates that it is expressed in a number of gastrointestinal and extragastrointestinal tissues.

Hairpin formation within the enhancer region of the human enkephalin gene.

The 3',5'-cyclic adenosine monophosphate (cAMP)-inducible enhancer of the human enkephalin gene is located within an imperfect palindrome of 23 base pairs. We have found that a 23-base-pair oligonucleotide duplex containing the enhancer undergoes a reversible conformational transition from the duplex to two individual hairpin structures each formed from one strand of the duplex. Each individual hairpin forms with mismatched base pairs, one containing two GT pairs and the other containing two AC pairs. The conformational transition is stabilized by proton transfer to the hairpin containing AC mismatched pairs. The unique physical and thermodynamic properties of the enkephalin enhancer DNA suggest a model in which DNA secondary structure within the enhancer region plays an active role in cAMP-inducible activation of the human enkephalin gene via formation of cruciform structures.

Regulation of expression of opioid peptide genes.

In the past three years it has been shown by recombinant DNA approaches that there are at least three different genes that code for opioid peptides. The basic structures of the three polyprotein precursor molecules from which bioactive opioid peptides are derived are remarkably similar. There are also similarities in the structure of the genes that code for these precursor molecules. Using immunological techniques, it has been shown that the levels of the opioid peptides can be regulated by altering the rates of protein processing or secretion. Recently, complementary DNA clones of the opioid peptide precursor molecules have been used as hybridization probes to determine that regulation also occurs at the level of gene expression (transcription of the opioid peptide genes).