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RNA Editing in the Wilms' Tumor Susceptibility Gene, Wtl

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RNA Editing in the Wilms' Tumor Susceptibility Gene, Wtl Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press RNA editing in the Wilms' tumor susceptibility gene, WTl Prem Mohini Sharma/ Marianne Bowman/ Stephen L. Madden/ Frank J. Rauscher III/ and Saraswati Sukumar^'^ ^The Salk Institute for Biological Studies, La Jolla, California 92037 USA, ^ The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 USA Rat kidney WTl cDNAs contain either a thymidine or a cytosine residue at position 839. Genomic WTl DNA contains only T*^'. To explain these results, we propose the WTl transcript undergoes RNA editing in which U*^' is converted to C, resulting in the replacement of leucine 280 in WTl by proline. RNA editing at the same nucleotide was observed in WTl cDNAs from human testis. In functional assays, the WTl-leucine^*** polypeptide repressed the EGR-1 promoter in in vitro assays -30% more efficiently than WTl-proline. Edited WTl-C*^^ mRNA was barely detectable in neonatal kidney, whereas adult rat kidneys contained both U*^' and C*^'-WT1 mRNA, suggesting a role for the two protein isoforms in growth and differentiation. [Key Words: RNA editing; WTl; rat; human; developmental regulation] Received November 5, 1993; revised version accepted February 1, 1994. Wilms' tumor (WT) or nephroblastoma, a common pedi­ 5, in the region proximal to the zinc finger domain, and atric, solid malignancy, accounts for 8% of childhood 3 amino acids (lysine, threonine, and serine) between the cancers. It occurs in both sporadic and familial forms third and the fourth zinc fingers. The least common is (Haber and Buckler 1992; Haber and Housman 1992; the transcript lacking both splices (Haber et al. 1991; P. Slater and Mannen 1992). There is considerable hetero­ Sharma et al., unpubl.). WTl acts as a transcriptional geneity in the pathology of WTs, and several genes have regulator, a function consistent with the presence of four been implicated in its etiology (Haber and Buckler 1992; ZF motifs in the carboxy-terminal region (Call et al. Haber and Housman 1992; Slater and Mannen 1992). 1990; Gessler et al. 1990) and a negatively charged pro- One of the genes, WTl, located on human chromosome line/glutamine-rich trans-regulatory domain (Mitchell llpl3, is categorized as a tumor susceptibility gene for and Tjian 1989) at the amino terminus. The four contig­ WT, as the loss of these sequences is associated with the uous zinc fingers of the cysteine-histidine {C2H2) class development of childhood malignancies of the kidney. recognize and bind to the same DNA sequences (5'- The complete cDNA sequence and the intron-exon or­ GCGGGGGCG-3') as the early growth response-1 (EGR- ganization of the WTl gene have been deduced (Call et 1) gene product (Rauscher et al. 1990). Other genomic al. 1990; Gessler et al. 1990; Sharma et al. 1992). The sequences, to which the alternately spliced forms of WTl gene contains 10 exons spanning 50 kb of DNA WTl protein bind, have been identified (Bickmore et al. (Gessler et al. 1990; Haber et al. 1991), which is ex­ 1992). Although DNA binding is mediated by the zinc pressed as a 3.0-kb mRNA in the rat (Sharma et al. 1992). finger domain, the amino-terminal domain of the WTl The open reading frame of WTl mRNA is 1725, 1719, protein functions as a repressor of transcription in tran­ and 1576 nucleotides in human, mouse, and rat respec­ sient transfection assays utilizing the promoter se­ tively, and encodes a zinc finger protein of 52-54 kD quences of the EGR-1 (Madden et al. 1991), platelet-de­ with four zinc fingers (Zfs), which is expressed at high rived growth factor-A chain (PDGF-A) (Gashler et al. levels during kidney development. At least four different 1992; Wang et al. 1992), insulin-hke growth factor-2 WTl mRNAs are expressed, which reflect the presence (IGF-2) (Madden et al. 1991), and insulin-like growth fac­ or absence of an alternatively spliced exon 5, and three tor-1 receptor (IGF-IR) (Werner et al. 1993) genes. These codons in exon 9 (Pelletier et al. 1991). These several studies reinforced the anticipated function of WTl as a mRNAs generate proteins with altered DNA-binding tumor suppressor protein, targeting and repressing, spe­ specificities predictive of distinct physiological roles for cifically, positive regulators of cell growth. However, each protein (Madden et al. 1991; Bickmore et al. 1992). WTl may perform dual functions, as a transcriptional The alternative splice that is most abundant in the kid­ activator as well as a repressor. Recently, Wang and col­ ney contains 17 amino acids encoded by a separate exon leagues (Wang et al. 1993) performing experiments with the minimal promoter sequences of the PDGF-A chain ^Conesponding author. gene, have provided evidence that the amino-terminal 720 GENES & DEVELOPMENT 8:720-731 © 1994 by Cold Spring Harboi Laboratory Press ISSN 0890-9369/94 $5.00 Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press RNA editing in WTl region of WTl contains separate regulatory domains that Dawley rats. We amplified the region containing the function either to activate or suppress transcription. T -^ C conversion, in genomic DNA extracted from sev­ In the course of sequencing cDNAs for WTl in rats, we eral pairs of normal and tumorous kidneys, as well as observed two alternate forms of cDNAs, as judged by kidneys from six untreated rats by PCR. The PCR prod­ sequence differences at nucleotide 839. One form con­ ucts were screened for the presence of sequence variants tains CTC, coding for leucine 280, and the other form by digestion with Mnll and by single-stranded conforma­ contains CCC, encoding proline 280 (Sharma et al. 1992). tion polymorphism (SSCP) (Orita et al. 1989a,b) analysis. The data in this paper show that this difference occurs by On digestion with Mnll, the T^^^ yields 52-, 27-, and RNA editing. This RNA editing event is developmen- 13-bp fragments. Replacement of the T with C^^^ results tally regulated in rat kidney and testis. The importance in loss of one of the Mnll restriction sites, yielding 79- of these variant RNAs is underscored by the fact that and 13-bp fragments instead. SSCP analysis identifies they are evolutionarily conserved between rat and hu­ the base change in the WTl DNA as shifts in electro- man. Furthermore, the Leu/Pro dimorphism affects the phoretic mobility of single-stranded DNA on nondena- transcription repression function of the WTl protein. turing gels. The plus and minus strands of DNA carrying Our results suggest that RNA editing in WTl adds a new the T/A^^^ migrate slower than the ones carrying dimension to developmental stage-specific functions of the WTl protein. The results of our analyses were unexpected. None of the 26 genomic DNA samples showed the bands diag­ nostic of absence of the Mnll site or bands with altered gel migration reflective of the presence of a polymorphic Results nucleotide 839. cDNA samples from several of the same Single-stranded conformation polymorphism, tissues had previously been positive in both assays (data restriction cleavage, and sequencing analyses not shown). The results of a representative SSCP exper­ of PCR-amplified products reveal that WTl cDNA, iment, where the migration of single-stranded exon 6 but not genomic DNA, contains either aTor a C DNA and cDNA derived from five rat tissues (three nor­ at nucleotide 839 mal tissues and two kidney tumors), and an immortal­ ized rat embryo fibroblast cell line (Rat 2) is compared, On sequencing several cDNA clones from a rat testis are shown in Figure 1 A. Of the six cDNA/genomic DNA library, either a T or a C residue was found at nucleotide pairs, mobility shifts were observed in cDNA-derived 839 of rat WTl (Sharma et al. 1992). This change results PCR fragments from Rat 2 cell line, a carcinogen-in­ in the loss of a Mnll restriction cleavage site. To deter­ duced kidney tumor, 1166-1, and adult kidney. No shifts mine whether the T/C difference at 839 is a genetic poly­ were observed in fragments from the kidney tumor 1972- morphism, we examined DNA and RNA from Sprague- 8, adult liver, and newborn kidney. No mobility shifts A A/ 9> > T C G A BamHI EcoRI Pst I »* # ^* # ^ o^c># 1 a b a b a b' kb rtrtrts^rtfirfd; -23.0 -9.4 -6.6 -4.4 T839 <JC839 -2.3 -2.0 exon 6 SSCP Figure 1. (A)The T®^^ —>C conversion occurs in the WTl transcript but is absent in the WTl gene of both normal and tumor tissues. SSCP analysis of RT-PCR-amplified WTl cDNA and DNA pairs in the region of exon 6 from Rat 2 cells, NMU-induced kidney tumors, 1972-8 and 1166-1, adult kidney (ADK), adult liver (ADLi), and newbom kidney (NBK). The ^^P-labeled, PCR-generated 92-bp DNA fragments (exon 6, using 5451 and 5449 primers) from cDNA [a] and DNA {b] were electrophoresed under nondenaturing conditions as described in Materials and methods. Plasmid DNA containing cloned B2 cDNA fragments with either a T (CTC) or C (CCC) at nucleotide 839 served as source of controls for PCR of exon 6 and mobility shifts in PCR-SSCP. (B] The nucleotide sequence of the adult kidney DNA {left] and cDNA [right] in the region of exon 6 showing the T -^ C substitution at position 839 in the cDNA. The single-base change is marked by an asterisk (*) and has been confirmed by sequencing the complementary strand. (C) Southern analysis of WTl sequences in normal rat kidney.
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