Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press A novel translational repressor mRNA is edited extensively in livers containing tumors caused by the transgene expression of the apoB mRNA-editing enzyme Shinya Yamanaka/ Karen S. Poksay,^ Kay S. Arnold/ and Thomas L. Innerarity 2-5 ^Gladstone Institute of Cardiovascular Disease, -^Cardiovascular Research Institute, and "^Department of Pathology, University of California, San Francisco, California 94141-9100 USA Transgene expression of the apoUpoprotein B mRNA-editing enzyme (APOBEC-1) causes dysplasia and carcinoma in mouse and rabbit livers. Using a modified differential display technique, we identified a novel mRNA (NATl for novel APOBEC-1 target no. 1) that is extensively edited at multiple sites in these livers. The aberrant editing alters encoded amino acids, creates stop codons, and results in markedly reduced levels of the NATl protein in transgenic mouse livers. NATl is expressed ubiquitously and is extraordinarily conserved among species. It has homology to the carboxy-terminal portion of the eukaryotic translation initiation factor (elF) 4G that binds eIF4A and eIF4E to form eIF4F. NATl binds eIF4A but not eIF4E and inhibits both cap-dependent and cap-independent translation. NATl is likely to be a fundamental translational repressor, and its aberrant editing could contribute to the potent oncogenesis induced by overexpression of APOBEC-1. [Key Words: APOBEC-1; RNA editing; translation repressor; transgenic mice] Received October 28, 1996; revised version accepted December 18, 1996. ApoUpoprotein B (apoB) mRNA editing is the deamination and tissues (Driscoll and Zhang 1994; Giannoni et al. of a specific cytidine (nucleotide 6666) to form uridine in 1994; Yamanaka et al. 1994). Thus, APOBEC-1 appears the 14-kb apoB mRNA (Chen et al. 1987; Powell et al. to be part of a multiprotein complex (Harris et al. 1993; 1987). This deamination changes a glutamine codon Giannoni et al. 1994). (CAA) to a translation termination codon (UAA) and re­ Quite unexpectedly, we found that the overexpression sults in the formation of an apoB protein (apoB48) con­ of APOBEC-1 causes dysplasia and hepatocellular carci­ sisting of the amino-terminal 48% of the full-length ge- noma in transgenic mouse livers (Yamanaka et al. 1995). nomically encoded apoB (apoBlOO) (Scott 1995; Innerar­ It also induced hepatic dysplasia in a transgenic rabbit ity et al. 1996). ApoB mRNA is edited in the small founder. Overexpression of APOBEC-1 resulted in the intestines of mammalian species as well as in the livers development of hepatocellular carcinoma as early as 21 of some mammals such as mice and rats (Greeve et al. days after birth in transgenic mice. This potent oncogen­ 1993). The enzyme APOBEC-1 (apoB mRNA-editing esis was not attributable to transforming elements in the catalytic subunit polypeptide 1) that catalyzes the cyti­ vector, insertion effects of transgenes, or the increased dine deamination has been cloned (Teng et al. 1993). formation of the apoB48 protein. We hypothesized that APOBEC-1 possesses RNA-binding activity (Anant et al. the oncogenesis is a result of the aberrant editing of other 1995; Navaratnam et al. 1995) and cytidine deaminase mRNA(s) encoding protein(s) with important cellular activity (Navaratnam et al. 1993) but is inactive without function(s). If this hypothesis is correct, the identifica­ the addition of protein extracts that presumably contain tion of other target mRNAs of overexpressed APOBEC-1 missing auxiliary or complementary factors necessary could lead to the discovery of molecules important in for editing. These unidentified auxiliary proteins are regulating cell growth. widely but not ubiquitously distributed in many organs To identify other targets of APOBEC-1, we initially took a candidate mRNA approach (Yamanaka et al. 1995, 1996). Mutagenesis studies suggested that the sequence specificity of apoB mRNA editing is provided by a moor­ ^Present address: Department of Pharmacology, Osaka City University ing sequence—an 11-nucleotide sequence motif located Medical School, Osaka 545, Japan. ^Corresponding author. five nucleotides downstream from the edited cytidine E-MAIL [email protected]; FAX (415) 285-5632. ((-6666| -j^ ^pQg mRNA (Shah et al. 1991; Backus et al. GENES & DEVELOPMENT 11:321-333 © 1997 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/97 $5.00 321 Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Yamanaka et al. 1994). In vitro, the mooring sequence was sufficient to sequence and amplified the 92-bp sequence from control induce editing of cytidines four to six nucleotides up­ mouse liver RNA, transgenic mouse liver RNA, and stream in chimeric RNA (Driscoll et al. 1993; Backus mouse genomic DNA. When sequenced, the PCR prod­ and Smith 1994). We searched genomic databases for ucts from control mouse liver RNA and the genomic other mRNAs containing the mooring sequence and DNA were identical to the human sequence. In contrast, found six mRNA candidates that have a mooring-like the PCR products from the APOBEC-1 transgenic mouse sequence as well as cytidines four to six nucleotides up­ liver RNA had thymidines in the place of several cyti­ stream. The upstream cytidines of these mRNAs were dines, as in the DNA from differential display, further examined for editing. In the APOBEC-1 transgenic suggesting that these cytidines are edited in the trans­ mouse livers, only one of these six candidates [a tyrosine genic mouse livers. Primer extension analysis also con­ kinase mRNA (Mano et al. 1990)] showed detectable ed­ firmed the editing of multiple cytidines in both trans­ iting (-1%), which did not change the encoded amino genic mouse livers and in the transgenic rabbit founder acid. No cytidines in any of the six candidate mRNAs that overexpressed rabbit APOBEC-1 in their livers and were edited in control mouse livers. This candidate ap­ developed severe hepatic dysplasia (Fig. IC). This mRNA proach suggests that the pathological editing by overex- has been designated NATl. pressed APOBEC-1 remains selective and requires other Northern blot analyses revealed that NATl is ex­ unidentified element(s) in addition to the mooring se­ pressed at high levels in adult human heart, brain, pla­ quence. It became apparent that better approaches would centa, lung, liver, skeletal muscle, kidney, and pancreas be required to identify other target mRNAs, especially (Fig. ID), as well as in spleen, thymus, prostate, testis, those that are not in the genomic databases. ovary, small intestine, colon, and peripheral blood leu­ In this study, we used a modified differential display kocytes (data not shown). NATl is also expressed in fetal technique (Liang and Pardee 1992) to search for edited human brain, lung, liver, and kidney (data not shown). mRNAs. We identified, cloned, and characterized a All human cell lines examined expressed NATl at ap­ novel mRNA that is extensively edited in transgenic proximately the same levels, including HL-60 (promy- mouse and rabbit livers. This mRNA, NATl (novel elocytic leukemia), HeLa S3, K-562 (chronic myeloge­ APOBEC-1 target 1), encodes a protein that appears to be nous leukemia), MOLT-4 (lymphoblastic leukemia), Raji involved in the regulation of translation initiation. In (Burkitt's lymphoma), SW480 (colorectal adenocarci­ addition, comparison of apoB mRNA and NATl se­ noma), A549 (lung carcinoma), and G361 (melanoma) quences gave us new insights into the mechanism of (data not shown). Southern blot analysis (Fig. IE), in APOBEC-1-mediated mRNA editing. which human EST cDNA was used as a probe under highly stringent conditions, demonstrated that the NATl sequence is highly conserved among mammalian Results species (human, monkey, rat, mouse, dog, bovine, and rabbit). A weaker band present in chicken suggested the Identification of NATl existence of a NATl homolog in this species. No band We modified the differential display technique to selec­ was detected in yeast. The high degree of conservation tively amplify mRNAs that were edited in the livers of and the ubiquitous distribution of the mRNA suggest transgenic mice overexpressing APOBEC-1. Primers that NATl has critical functions in cellular physiology. (mooring primers) were designed that consisted of a se­ quence complementary to the mooring sequence, three to five degenerate nucleotides, and an adenosine at the 3' end (Fig. lA). Cytidines four to six nucleotides upstream Cloning of the full-length NATl cDNA of the mooring sequence can be edited in vitro (Driscoll We sequenced the longest clone (2.4 kb) of five human et al. 1993; Backus and Smith 1994). These mooring EST clones derived from NATl mRNA. That it was not primers served as antisense primers for cDNA synthesis a full-length clone was revealed when Northern blot and PCR instead of the anchored oligo-dT primers usu­ analysis demonstrated a 4-kb transcript (Fig. ID). Using ally used in the differential display procedure (Liang and 5'-RACE-PCR (Frohman et al. 1988), we obtained a full- Pardee 1992). Using this modified technique, we identi­ length cDNA of human NATl (3.8 kb). Mouse and rabbit fied differentially amplified bands on agarose gels (Fig. NATl full-length cDNAs were obtained by RT-PCR IB) from which DNAs were eluted, subcloned, and se­ with primers based on the human sequence (Fig. 2). At quenced. A search of genomic databases revealed that the nucleotide level, human and rabbit sequences are two of the three clones shown in Figure IB (clones 3-13 95.5% and 94.8% identical, respectively, to the mouse and 3-13') were nearly identical to each other and to sequences. The 3' sequence of NATl mRNA contains several human expressed sequence tags (ESTs) (Adams et five sequence motifs that are only one or two nucleotides al. 1992). Importantly, the mouse sequences derived different from the apoB mRNA mooring sequence (Figs.