Proc. Nati. Acad. Sci. USA Vol. 84, pp. 7261-7265, October 1987 Medical Sciences Characterization of proopiomelanocortin transcripts in human nonpituitary tissues (testis/RNA blot hybridization/Si nuclease mapping/transcription) THIERRY LACAZE-MASMONTEIL*, YVES DE KEYZERt, JEAN-PIERRE LUTONt, AXEL KAHN*, AND XAVIER BERTAGNAtt *Unit6 de recherches en gdndtique et pathologie mol~culaires, Institut National de la Sante et de la Recherche Medicale Unit6 U-129; and tCentre de Recherches sur les Maladies Endocriniennes, H6pital Cochin, 27 rue du Faubourg St Jacques, Paris 75014, France Communicated by Alfred Jost, June 1, 1987 (received for review February 23, 1987) ABSTRACT Proopiomelanocortin (POMC), the precursor indicated that POMC processing in the rat testis is similar to to adrenocorticotropic hormone and other related peptides, that in the pituitary intermediate lobe cell (9). was originally identified in the corticotropic cell. Recent POMC mRNA was subsequently identified in rat and evidence shows that POMC products are also normally present mouse testis by blot hybridization analysis (10, 11) and in a variety of nonpituitary tissues. To investigate this phe- precisely located in the mouse Leydig cell by in situ hybrid- nomenon in humans we looked for the presence and charac- ization (12), providing the best evidence for POMC gene teristics of POMC transcripts in various adult tissues. Blot expression in this tissue. Surprisingly, the POMC transcripts hybridization analysis of normal adrenal, thymus, and testis were quite smaller than those in the pituitary (11), and the RNAs revealed a small RNA species approximately 400 nucle- peptide content appeared extremely low for the amount of otides shorter than the 1200-nucleotide pituitary species. Prim- POMC mRNA in comparison to what is observed in other er extension and S1 nuclease mapping studies showed that this tissues (9). small RNA lacked exon 1 and exon 2 of the gene, and it In this report we have identified and characterized the corresponded to a set ofat least six molecules starting 41 to 162 POMC transcripts in normal human nonpituitary tissues, nucleotides downstream from the 5' end ofexon 3. These RNAs mainly in the testis. Most of these transcripts are truncated appear to result from heterogeneous transcription initiation molecules lacking the 5' end of the coding region. On these sites presumably under the control of "GC box" promoter grounds we propose an explanation for the small amount of sequences located in the 3' end of intron 2. They cannot encode all the POMC products, which are nevertheless present in a complete POMC molecule, and the only truncated POMC these tissues. molecules that could be translated would lack a signal peptide necessary for membrane translocation and precursor process- MATERIALS AND METHODS ing. The use of highly sensitive S1 nuclease mapping techniques Isolation of RNA. Normal human pituitaries and nonpitu- with uniformly labeled single-stranded DNA probes allowed itary tissues were obtained at autopsy or as a by-product of the detection of a small but definite amount of the "normal," surgery. Total RNA was extracted by the guanidium chloride 1200-nucleotide, mRNA species. It is suggested that it is this procedure (13) as modified in our laboratory (14). Poly(A)- POMC mRNA that is responsible for the local production ofall enriched fractions were isolated by oligo(dT)-cellulose chro- the POMC peptides. matography (15), with a yield of about 5% (wt/wt). Blot Hybridization Analysis. RNA samples were denatured Proopiomelanocortin (POMG) is a polypeptide molecule that with 10 mM methylmercuric hydroxide (16), fractionated on was identified as a precursor to adrenocorticotropic hormone denaturing 2% agarose gels, blotted onto nylon filters (ACTH) in corticotropic cells (1, §). Its primary structure was (GeneScreenPlus from New England Nuclear), and hybrid- established after the cDNA sequence was obtained from beef ized with single-stranded DNA probes prepared from phage pituitary mRNA (2), and the gene structure was unravelled in M13 subclones containing POMC genomic fragments and many species, including humans (3-6). These data provided encompassing each of the three exons of the human gene. the molecular basis for the mechanism of POMC processing Probe Synthesis. Single-stranded DNA probes of a defined which, besides ACTH, releases a number of fragments with size, complementary to POMC genomic fragments subcloned potential biological activity such as the 16-kDa fragment, the in M13 vector, were obtained by extension from the M13 melanocyte-stimulating hormones, the lipotropins, and the universal primer (17). Two probes were complementary to endorphins (7). the coding region of the third exon: pHOX 3A, 707 bases (b) Numerous reports have recently shown that POMC pep- long, encompassed 657 b of the gene; pHOX 3C, 155 b long, tides are also normally present in extracts of various was generated by extension from the 5'-CCCGCTGAGAC- nonpituitary tissues including brain, gastrointestinal tract, GTCCTC-3' oligonucleotide complementary to the third placenta, and male and female gonads (7). One of the most exon and included 8 b of vector sequence at its 3' end. This extensively studied tissues is the testis. Immunocytochemi- POMC-specific primer was also labeled with polynucleotide cal studies with antisera directed against the y3-melanocyte- kinase to generate a 5'-end-labeled probe. stimulating hormone, ACTH, and p-endorphin of the precur- S1 Nuclease Mapping. The technique was performed as sor have located immunostainable POMC products in Leydig described previously (18). Briefly, uniformly or 5'-end-la- cells of many species (8). The molecular forms of these products, as assessed by gel exclusion and HPLC techniques, Abbreviations: ACTH, adrenocorticotropic hormone; POMC, pro- opiomelanocortin; b, base(s); U, units. tTo whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page charge §Orth, D. N., Nicholson, W. E., Shapiro, M. & Byyny, R., Program payment. This article must therefore be hereby marked "advertisement" ofthe Endocrine Society, 52nd Meeting, June 1970, St. Louis, p. 140 in accordance with 18 U.S.C. §1734 solely to indicate this fact. (abstr.). 7261 Downloaded by guest on September 29, 2021 7262 Medical Sciences: Lacaze-Masmonteil et al. Proc. Natl. Acad. Sci. USA 84 (1987) beled single-stranded probes were hybridized to RNA in 10 Characterization of the Small POMC Transcripts by S1 ,ul of 40 mM 1,4-piperazinediethanesulfonic acid (Pipes), pH Nuclease Mapping and Primer Extension Studies. Uniformly 6.4/80% (vol/vol) formamide/0.4 M NaCi at 480C (560C with labeled pHOX 3A probe was hybridized with testis poly(A)+ pHOX 3A probe) for 12 hr. Then the heteroduplexes were RNA and subjected to S1 nuclease digestion, and the pro- digested in 200 ,4 of 250 mM NaCI/50 mM sodium acetate, tected fragments were separated by electrophoresis (Fig. 2). pH 4.6/2.5 mM ZnSO4 containing S1 nuclease at 5000 units Several bands were detected. The slowest one was 657 b (U) (as defined by the supplier, Bethesda Research Labora- long, corresponding to the entire POMC-specific part of the tories) per ml for 2 hr at 250C. After extraction with phenol probe, and was similarly protected in the pituitary (Fig. 2, and precipitation with ethanol, protected fragments were lane 3); smaller fragments, at least three, were also detected, separated on a 6% (4% for pHOX 3A-generated fragments) with a size of approximately 600 b. To better characterize polyacrylamide/7 M urea gel. these protected fragments we used a smaller, 155-b single- Primer Extension. RNA samples were first denatured 20 stranded DNA probe (pHOX 3C) generated from a synthetic min in 90% (vol/vol) dimethyl sulfoxide at 450C and precip- 17-mer primer complementary to the position 7013-7029 itated with ethanol. Then they were hybridized to the fragment of the gene (Fig. 3C). S1 nuclease mapping studies 5'-labeled POMC primer in 10 ,lI of 100 mM Tris-HCI, pH were performed on testis, thymus, and pituitary RNA, using 7.5/150mM KCI/60 mM MgCl2 at 65°C for 15 min and at 42°C first a uniformly labeled pHOX 3C probe (Fig. 3A); five for 1 hr. Each sample was adjusted to 20 ,4 of 50 mM protected fragments were detected. The slowest one was 147 Tris HCI, pH 7.5/75 mM KCI/3 mM MgCl2/2 mM sodium b long, corresponding to the entire POMC-specific part ofthe pyrophosphate/1 mM dNTP with 40 U (as defined by the probe, and was similarly protected in the pituitary (Fig. 3A, supplier) of the Moloney murine leukemia virus reverse lane 4). The four others were specific for the nonpituitary transcriptase (Bethesda Research Laboratories) and extend- tissues and were more abundant in the testis than in the ed for 1 hr at 42°C. After extraction with phenol and thymus (Fig. 3A, lane 5). When the same studies were precipitation with ethanol, the extension products were performed with the 5'-end-labeled pHOX 3C probe, the same separated on a 6% polyacrylamide/7 M urea gel. protected bands were again observed (data not shown), confirming that the heterogeneity of these POMC transcripts RESULTS resulted from variable 5' extremities. In this latter experi- ment, similar intensities ofthe signals also indicated that each Identification of the Small POMC Transcripts in Human POMC transcript was present in approximately equal Tissues. Total or poly(A)+ RNA prepared from normal amounts. adrenal, thymus, testis, and pituitary was subjected to blot To precisely locate the 5' end of the transcript protecting hybridization analysis. The hybridization was performed all the POMC sequence of pHOX 3C, primer extension with a 707-b single-stranded DNA probe encompassing most studies were performed with the synthetic 17-mer primer of the third exon coding region (pHOX 3A). POMW tran- (Fig. 3B). The longest extension product was 157 b long, scripts were detected in the testis and thymus (Fig.