Proc. Nati. Acad. Sci. USA Vol. 85, pp. 4218-4222, June 1988 Biochemistry Primary structure of the human follistatin precursor and its genomic organization (ovary/testis/pituitary/prehormone/alternative splicing) SHUNICHI SHIMASAKI*, MAKOTO KOGA*, FREDERICK ESCHt, KAREN COOKSEY*, MALUZ MERCADO*, ANN KOBA*, NAOTO UENO*, SHAO-YAO YING*, NICHOLAS LING*, AND ROGER GUILLEMIN* *Laboratories for Neuroendocrinology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037; and tAthena Neurosciences, Inc., 887-D Industrial Road, San Carlos, CA 94070 Contributed by Roger Guillemin, February 23, 1988 ABSTRACT Follistatin is a single-chain gonadal protein characterized porcine inhibins A and B. By use ofthe porcine that specifically inhibits follicle-stimulating hormone release. cDNA as a probe, we report herein the cloning and sequenc- By use ofthe recently characterized porcine follistatin cDNA as ing of the human follistatin precursor as well as the deter- a probe to screen a human testis cDNA library and a genomic mination of its genomic organizationA library, the structure of the complete human follistatin pre- cursor as well as its genomic organization have been deter- mined. Three of eight cDNA clones that were sequenced MATERIALS AND METHODS predicted a precursor with 344 amino acids, whereas the Cloning and DNA Sequencing. An oligo(dT)-primed human remaining five cDNA clones encoded a 317 amino acid precur- testicular cDNA library constructed with the vector Agtll sor, resulting from alternative splicing ofthe precursor mRNA. was purchased from Clontech (Palo Alto, CA). The mRNA Mature follistatins contain four contiguous domains that are used to produce the library was derived from the testis of a encoded by precisely separated exons; three of the domains are healthy 50-year-old man. The library was screened with a highly similar to each other, as well as to human epidermal porcine cDNA probe encoding the follistatin precursor. growth factor and human pancreatic secretory trypsin inhib- Hybridization was done in 1 M NaCI/1% NaDodSO4/10% itor. The genomic organization of the human follistatin is dextran sulfate/50% (vol/vol) formamide containing 0.2 mg similar to that of the human epidermal growth factor gene and of denatured salmon sperm DNA per ml at 37°C for 15 hr. thus supports the notion of exon shuffling during evolution. Filters were washed at 50°C for 10 min with 15 mM NaCl/1.5 mM sodium citrate/0.19o NaDodSO4. Twelve positive clones The granulosa cells of the ovary secrete into the follicular were detected that were divided into four groups according to fluid many proteins that can modify the secretion ofpituitary the length of the inserted cDNA fragment; the 1.00-kilobase follicle-stimulating hormone (FSH). Two such proteins, (kb) group ofclones HTF 101, 103, and 108; the 1.50-kb group named inhibins A and B, which specifically inhibit the containing only clone HTF 109, the 1.40-kb group of clones secretion of pituitary FSH, have been isolated and charac- HTF 102, 104, 105, 107, and 110-112; and the 1.85-kb group terized from porcine (1-3) and bovine (4, 5) follicular fluid. containing only clone HTF 106. Eight clones (HTF 102 and The inhibins are heterodimeric proteins of M, 32,000, com- HTF 106-112) were subcloned into the M13mpl9 vector and posed of a common glycosylated a subunit of Mr 18,000 and sequenced in both directions by the dideoxy chain- one of two similar , subunits of Mr 14,000 (1, 6). In addition, termination method (13) with synthetic primers based on the homo- and heterodimers ofthe /3 subunits ofinhibins, named porcine follistatin cDNA sequence (12). activins (7, 8) or FRP (9), have also been identified in porcine For the follistatin gene cloning, a human lymphocyte follicular fluid, which can specifically stimulate the secretion genomic library, constructed in the vector EMBL 3 with 15- of pituitary FSH. Throughout the purification of the inhibins to 20-kb DNA fragments produced by partial digestion with and activins, we consistently observed one side fraction that Mbo I, was purchased from Stratagene (San Diego, CA). It also has FSH-release inhibitory activity and appeared unre- was screened with the same porcine cDNA probe as above. lated to the inhibins. The fractions containing this activity Three positive clones (HFG 101-103) were identified. The have been purified to homogeneity to yield two glycosylated insert fragments of the follistatin gene clones were excised to single-chain proteins of Mr 35,000 and 32,000, respectively the appropriate length, subcloned into pUC18 or M13mpl9 (10). The two proteins have similar amino acid compositions vectors, and sequenced in both directions by the same and identical amino-terminal amino acid sequences and were method as above. Compressed sequence ladders were clar- named follistatins. Subsequently, three proteins with FSH ified by the addition ofEscherichia coli single-strand binding release-inhibitory activity and having the same amino- protein to the sequencing reaction (Pharmacia). terminal amino acid sequence as porcine follistatins were Ovarian RNA Preparation and RNA Blot Hybridization. isolated from bovine follicular fluid by Robertson et al. (11). Human ovaries were obtained from a patient with radical By use of amino acid sequence information derived from endometriosis. The tissues were quick frozen in liquid nitro- trypsin digestion ofthe Mr 35,000 protein, two populations of - cDNA clones encoding, respectively, a follistatin precursor gen immediately after excision and stored at 80°C until of 344 amino acids or its carboxyl-terminal truncated form ready for RNA preparation. Total RNA was prepared by the with 317 residues were identified from a porcine ovarian conventional guanidine isothiocyanate method (14) and cDNA library (12). The deduced precursor sequences bear no homology with the a, PA, and PB chains of the previously Abbreviations: FSH, follicle-stimulating hormone; EGF, epidermal growth factor; preFS317 and preFS344, follistatin precursors of 317 and 344 amino acids, respectively. The publication costs of this article were defrayed in part by page charge $The sequence reported in this paper is being deposited in the payment. This article must therefore be hereby marked "advertisement" EMBL/GenBank data base (IntelliGenetics, Mountain View, CA, in accordance with 18 U.S.C. §1734 solely to indicate this fact. and Eur. Mol. Biol. Lab., Heidelberg) (accession no. J03771). Downloaded by guest on September 30, 2021 4218 Biochemistry: Shimasaki et al. Proc. Natl. Acad. Sci. USA 85 (1988) 4219 poly(A)+ RNA was selected by oligo(dT)-cellulose column To further delineate the origin of the two precursors, we chromatography. have cloned and sequenced the follistatin gene from a human Five micrograms of poly(A)+ RNA was electrophoresed genomic library. From 1 million phage plaques, three positive on a formaldehyde-agarose gel and then transferred onto a clones, HFG 101, HFG 102, and HFG 103 (Fig. ld), were nylon membrane filter. Hybridization with the 32P-labeled- obtained by hybridization with the porcine cDNA probe. HTF 109 insert fragment was done under the same conditions Restriction mapping and nucleotide-sequence analyses re- as those that were used for the plaque screening. The filters vealed that clone HFG 102, which is 19.1 kb in length, were washed with vigorous agitation in 300 mM NaCl/30 mM encodes nearly the whole human follistatin gene containing sodium citrate/0.1% NaDodSO4 for 15 min at room temper- five exons. These five exons encode, respectively, the signal ature and then incubated in 15 mM NaCl/1.5 mM sodium sequence and the first four domains (I-IV) of follistatin. citrate/0.1% NaDodSO4 for 1 hr at 650C. Autoradiography Clone HFG 103, which is 14.6 kb long, contains only the first was performed on Kodak XAR-5 film with an intensifying exon encoding the signal sequence, whereas the 15.0-kb screen for 6 hr at - 80'C. clone HFG 101 contains only the last exon (V) encoding the extra 27 carboxyl-terminal residues of preFS344. The 5'- terminal sequence of HFG 101 could not be merged with the RESULTS AND DISCUSSION 3'-end of HFG 102 to yield the complete gene sequence. From 8.2 x 105 phage plaques of an adult human testicular Nevertheless, the overall genomic organization of human Agtll cDNA library, 12 positive clones were obtained by follistatin can be postulated to be composed of a DNA hybridization with a porcine follistatin cDNA probe, and 8 sequence that is >5 kb in length with six exons interrupted by positive clones were selected for sequencing. From the five introns (Fig. ic). Aside from the first exon, which deduced nucleotide sequences, two populations of cDNAs encodes the signal sequence, the following four exons en- that differ in their 3'-terminal coding regions were detected. coding domains I-IV are of approximately equal size. The One population consists of three clones (HTF 106, HTF 108, first four introns, where sequences have been determined and HTF completely, contain 2009, 430, 346, and 702 base pairs, 109, Fig. la) that encode a follistatin precursor of respectively, all of which possess the consensus 5'-GT... 344 amino acids (preFS344), whereas the other consists of AG-3' donor and acceptor splicing sites (Fig. 2). The exact five clones of the same length (HTF 102, HTF 107, and HTF length of the last intron could not be determined precisely. 110-112, Fig. lb) whose open reading frame encodes an Fig. 2 shows the composite cDNA and the corresponding identical precursor that lacks 3 amino acids at the amino amino acid sequence of the follistatin precursor as well as the terminal and 27 residues at the carboxyl terminal of five intron sequences in the follistatin gene. The upper preFS344. Shortage of the three amino-terminal amino acids portion of the figure (nucleotides 1-979) presents the com- in the latter precursor may be caused by termination of the plete cDNA sequence encoding the follistatin precursor of reverse transcription at that position during cDNA synthesis.