Proc. Natl. Acad. Sci. USA Vol. 90, pp. 537-541, January 1993 Genetics Conserved nucleotide sequences in the open reading frame and 3' untranslated region of selenoprotein P mRNA (selenium/UGA codon/selenocysteine/mRNA secondary structure) KRISTINA E. HILL*t, R. STEPHEN LLOYDtt, AND RAYMOND F. BURK*t Division of Gastroenterology, Departments of *Medicine and *Biochemistry; and tCenter in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232 Communicated by William J. Darby, October 6, 1992 (receivedfor review May 7, 1992) ABSTRACT Rat liver selenoprotein P contains 10 seleno- provide insight into selenocysteine incorporation. The pres- cysteine residues in its primary structure (deduced). It is the ent study examines the cDNAs of human and rat liver only selenoprotein characterized to date that has more than one selenoprotein P.§ It compares the 3'utr between the human selenocysteine residue. Selenoprotein P cDNA has been cloned and rat cDNAs and analyzes the 3'utr for clues to the from human liver and heart cDNA libraries and sequenced. mechanism of incorporation of multiple selenocysteine res- The open reading frames are identical and contain a signal idues. peptide, indicating that the protein is secreted by both organs and is therefore not exclusively produced in the liver. Ten selenocysteine residues (deduced) are present. Comparison of MATERIALS AND METHODS the open reading frame of the human cDNA with the rat cDNA Selection ofHuman Selenoprotein P cDNA Clones. The open reveals a 69% identity of the nucleotide sequence and 72% reading frame portion of the rat selenoprotein P cDNA clone identity ofthe deduced amino acid sequence. Two regions in the 16C1 was obtained from the cDNA by digestion with EcoRI 3' untranslated portion have high conservation between human and rat. Each of these regions contains a predicted stable and Xba I. This insert was purified by agarose gel electro- stem-loop structure similar to the single stemloop structures phoresis and electroelution onto an NA45 DEAE membrane reported in 3' untranslated regions of type I iodothyronine (Schleicher & Schuell). The isolated 16C1 open reading frame 5'-deiodinase and glutathione peroxidase. The stemloop struc- DNA was nick-translated with [a-32P]dCTP (NEN) and used ture of type I iodothyronine 5'-deiodinase has been shown to be to screen a UniZAP human liver library (Stratagene). The necessary for incorporation of the selenocysteine residue at the phage stock library was diluted and plated on Escherichia coli UGA codon. Because only two stem-oop structures are present XL-1 Blue cells (Stratagene) at a concentration of 5 x 104 in the 3' untranslated region of selenoprotein P mRNA, it can plaques per plate. The library was screened by using the be concluded that a separate stem-oop structure is not re- procedure described (5). A single plaque-purified cDNA quired for each selenocysteine residue. clone, 7C1, was purified and sequenced. A AZAP II human heart cDNA library (Stratagene) was Selenoproteins contain selenocysteine residues in their pri- screened in the same manner as described above. The open mary structure. Studies of bacterial and animal selenopro- reading frame portion of the 7C1 cDNA was obtained by teins have provided an outline of selenocysteine incorpo- digestion ofthe cDNA with EcoRI and Xba I. This insert was ration, but large gaps remain in our knowledge of this used to screen the heart library, and a single plaque-purified process. Selenocysteine is made from serine on a unique cDNA clone, 9A1, was sequenced. tRNA that recognizes certain UGA codons in the open Sequence Method and Strategy. Excision of 7C1 cDNA reading frame of mRNA (1). Recognition of UGA as coding pBluescript phagemid from the UniZAP phage or excision of for selenocysteine appears to be mediated by secondary the 9A1 cDNA pBluescript phagemid from the AZAP II phage structure of the mRNA. In bacteria, a stem-loop structure is was performed by incubating high titer phage stock and R408 present immediately downstream from the UGA (2). Disrup- helper phage with E. coli XL-1 Blue cells according to the tion of the stem or changes in single nucleotides on the loop Stratagene protocol. The recircularized phagemids were used abolish selenocysteine incorporation (2, 3). Thus, the stem- to infect new E. coli XL-1 Blue cells. The circular phagemids loop structure appears to interact with the ribosome directly were used to sequence the cDNA inserts. The Sanger dide- or indirectly to mediate selenocysteine incorporation. In oxy termination method was used to sequence double- animal systems, no stem-loop structure is found in the open stranded DNA. A r7Sequencing kit (Pharmacia LKB) was reading frame. However, a stable stem-loop structure has used to sequence the cDNA inserts. been described in the 3' untranslated region (3'utr) of mRNA The sequencing strategy used to sequence 7C1 cDNA is of the selenoproteins type I iodothyronine 5'-deiodinase shown in Fig. 1. The arrows represent the segment of cDNA (5'-deiodinase) and glutathione peroxidase (4). Mutants that sequenced and the direction of sequencing. The same strat- contain a disruption ofthis stem-loop structure in the 3'utr do egy was used to sequence the 9A1 cDNA. Oligomers, syn- not express 5'-deiodinase after transfection in JEG-3 or thesized on an Applied Biosystems 391 DNA synthesizer, COS-7 cells (4). Thus, a single stem-loop structure is nec- were used to prime the cDNA templates for all sequencing essary for the incorporation of the single selenocysteine reactions. residue present in these selenoproteins. Northern Analysis. Human kidney RNA was kindly pro- Selenoprotein P is the only selenoprotein that is known to vided by T. Daniel (Vanderbilt University), and human liver contain more than one selenocysteine residue in its primary RNA was kindly provided by E. Gillam (Vanderbilt Univer- structure (5). Because of this, study of it can be expected to Abbreviations: 3'utr, 3' untranslated region; 5'-deiodinase, type I The publication costs of this article were defrayed in part by page charge iodothyronine 5'-deiodinase. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. Z11793). 537 Downloaded by guest on September 26, 2021 538 Genetics: Hill et al. Proc. Natl. Acad. Sci. USA 90 (1993) 0 1000 2000 a b c d of g h I } k I m n p I' I I I 1I I I I I 1 10 2- 2 - 3 11.- 1 2 f 13 6 7 14 - 8 l -> 9 15 - FIG. 1. Sequencing strategy used for human selenoprotein P. The arrows represent the segment of cDNA sequenced and the direction of sequencing. T3 primer and M13 universal primer were used for numbers 1 and 15, respectively. All other sections were sequenced using oligomers specifically synthesized as primers. Unique restriction enzyme sites are designated on the scale, showing the base number. Restriction enzyme sites are indicated by letters: a, BstXI; b, Ava II; c, AlwNI; d, Bbv I and BspMI; e, Xmn I; f, Afl II; g, Gdi II and Eae I; h, Nde I; i, BspHI; j, HgiAI and Bsp 1286; k, BstEII; l, Sal I and Acc I; m, Hga I; n, Xba I; o, NspHI and Nsp 7524I; and p, Bsm I. sity). Northern analysis of total kidney and liver RNA was signal peptide. This indicates that the heart also makes performed as described (20). selenoprotein P and presumably secretes it. Computer Analysis. Secondary structure for RNA folding Northern Analysis for Selenoprotein P mRNA. Previous was predicted using MFOLD, version 2.0, written for the studies have shown that selenoprotein P is synthesized and Apple Macintosh by Don Gilbert (Biocomputing Office, secreted by the liver (5, 15). Evidence for selenoprotein P Biology Department, Indiana University). The program de- message in human liver and heart was obtained by isolation termines RNA secondary structure by free-energy minimi- of selenoprotein P cDNAs from these libraries. Direct evi- zation using the method of Zuker and coworkers (6-8). The dence for the presence of selenoprotein P message in human maximum nucleotide length for each folding determination kidney was obtained by Northern analysis (Fig. 3). The size allowed by the program was 300 bases. Sequential 300 base -of the message in liver and kidney was 2.2 kilobases. Levels sequences were folded initially, and then 150 bases of over- of mRNA appear to be an order of magnitude greater in liver lapping sequences were combined to give additional 300-base than in kidney because the intensity of the band detected sequences that were folded. The maximum size of an interior using 2 ,g of liver RNA was greater than that using 20 pug of loop was limited to 30 bases, and the maximum lopsidedness kidney RNA. The presence of selenoprotein P message in of an interior loop was limited to 30 bases. tissues other than the liver indicates that the protein is synthesized in several tissues and not just in the liver. Secondary Structure Predictions for Seenoprotei P mRNA RESULTS AND DISCUSSION and Comparison with mRNAs of Other"Selenoproteins. The Nucleotide Sequence, Deduced Amino Acid Sequence, and mechanism by which a selenocysteine residue is incorporated Selenocysteine Content of Human Selenoprotein P. The se- into protein is under active investigation. Studies with 5'- quence of the cDNA of human liver selenoprotein P with its deiodinase and glutathione peroxidase have shown that a part deduced amino acid sequence is shown in Fig. 2. Ten TGA ofthe 3'utr ofthe mRNA, which can form a stable stem-loop codons are present in the open reading frame of the cDNA. structure, is necessary for selenocysteine insertion (4).