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Hydrolytic nucleoside and nucleotide deamination, and genetic instability: a possible link between RNA-editing enzymes and cancer?
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Hydrolytic nucleoside and nucleotide deamination, and genetic instability: a possible link between RNA-editing enzymes and cancer? Shrikant Anant1 and Nicholas O. Davidson2 1
Department of Internal Medicine, Washington University School of Medicine, Saint Louis, MO 63110, USA Departments of Internal Medicine, and Molecular Biology and Pharmacology, Washington University School of Medicine, Saint Louis, MO 63110, USA 2
Post-transcriptional RNA editing generates novel gene products by changing the coding sequence of the transcript from that in the genome. Two classes of RNA editing exist in mammals, each of which involves an enzymatic deamination. These reactions have stringent sequence and structural requirements for their target RNAs, and each requires distinctive enzymatic machinery. Alterations in the expression or abundance of RNAediting factors produce unanticipated alterations in the processing or expression of RNAs, in some cases outside their physiological targets. Recent findings suggest that unregulated expression of the cytidine-deaminase gene family might lead to deamination of deoxycytidine nucleotides in DNA. Aberrant or dysregulated RNA editing, or altered expression of editing factors, might contribute to genomic instability in cancer. Several mechanisms exist by which organisms can expand their genetic repertoire without creating a new gene. These include the use of alternative promoters to create different mRNA transcripts from the same gene, and a variety of intranuclear, post-transcriptional processing and modification steps, such as splicing, editing and polyadenylation, by which the sequence of the primary transcript can be altered from that specified in the chromosomal gene. These various adaptations create a hierarchy of alternative coding templates or differentially processed transcripts, and thus permit considerable plasticity within the context of a single gene. In this article, we discuss the evidence that dysregulation of a specific class of post-transcriptional modification, RNA editing, might be associated with cancer. The underlying hypothesis is that aberrant RNA editing, mediated through alterations in the expression of one or more trans-acting factors, can lead to unanticipated alterations in the sequence and/or function of RNA transcripts beyond their canonical target. Recent findings also suggest that uncontrolled expression of protein components of the RNA-editing enzyme complexes might result in Corresponding author: Nicholas O. Davidson ([email protected]).
promiscuous nucleotide deamination in DNA. These aberrant processing events occur in human cancers and in experimental animal models, and emphasize the need to consider this type of gene regulation in the wider context of factors that contribute to genetic instability. RNA editing: overview and physiological regulation Post-transcriptional RNA editing is a process by which the nucleotide sequence of a nuclear transcript is changed from that encoded in the genomic template. This can alter the codon reading frame and amino acid sequence of the translation product, thereby changing the function of the gene product. In mammals, RNA editing occurs through base modification, via deamination of cytidine (C) to uridine (U) or of adenosine (A) to inosine (I), within particular nuclear mRNAs. A ! I RNA editing occurs on unspliced RNA templates and requires base-pairing between an exon and an adjacent intron in the targeted RNA [1 –3]. Inosine is recognized by the translational apparatus as guanosine, so the net effect of A ! I RNA editing is changes an A to a G in the reading frame, which in turn alters the sequence and function of the gene product [1 – 3]. Examples of mRNAs in which A ! I RNA editing occurs include those encoding the calcium-gated glutamate receptor GluR-B, hepatitis delta virus and the 5-hydroxytryptamine (5-HT2c) receptor [4]. The original and best-characterized example of C ! U editing is the mRNA encoding apolipoprotein B (apoB), in which a single, site-specific cytidine deamination introduces a translational termination codon (UAA) into the reading frame of the edited transcript, leading to production of the shortened isoform, apoB48 (Fig. 1a). This occurs exclusively in the human small intestine, where apoB48 is required for the absorption of dietary lipid [5]. ApoB RNA expressed in human liver is not edited and hepatic apoB RNA encodes a larger protein, apoB100. The carboxyl terminus of apoB100, which contains the binding domain for the low-density-lipoprotein receptor, is absent from the intestinal form (apoB48). Hence, C ! U RNA
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Legend APOBEC-1
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Holoenzyme complex for apoB RNA editing TRENDS in Molecular Medicine
Fig. 1. Site-specific deamination of cytidine (C) to uridine (U) (C ! U RNA editing) and the consequences of aberrant expression of the gene encoding apolipoprotein-BmRNA-editing-enzyme catalytic polypeptide 1 (APOBEC-1). (a) Under physiological conditions, C ! U RNA editing targets a single cytidine nucleotide in the nuclear transcript encoding intestinal apolipoprotein B (apoB). The C ! U editing enzyme complex includes a catalytic subunit APOBEC-1 (green), and an RNA-recognition subunit APOBEC-1 complementation factor (ACF) (pink). ACF binds APOBEC-1 and forms a higher-order complex within the nucleus. Several constraints could potentially act as regulatory points in this process. ACF is predominantly nuclear and its partitioning is distinct from that of APOBEC-1, which is predominantly cytoplasmic (i). APOBEC-1 binds to ACF as well as other auxiliary proteins [such as GRY-RBP (glycine-, arginine- and tyrosine-rich RNA-binding protein) and CUGBP2 (CUG-triplet-repeat RNA-binding protein 2)] (blue) in the cytoplasm (ii), and in the nucleus (iii). C ! U RNA editing of apoB requires an optimal nuclear stoichiometry of ACF and APOBEC-1 within the context of a holoenzyme (iv), which allows targeted cytidine deamination of a single base in nuclear apoB mRNA, resulting in the production of apoB48. (b) Aberrant regulation of APOBEC-1 expression leads to unanticipated changes in transcripts other than apoB. If APOBEC-1 is overexpressed (either forced transgenic or sporadic overexpression) (i), cytoplasmic transcripts containing AU-rich elements might be bound by APOBEC-1 (ii), causing alterations in their stability and/or translation (iii). In addition, increased abundance of APOBEC-1 could result in increased diffusion or transport into the nucleus (iv) where other mRNA targets might be susceptible to enzymatic deamination. These include the mRNAs encoding neurofibromatosis type 1 (NF1) (v) and NAT1 (vi). APOBEC-1 overexpression results in C ! U editing at a single site in NF1 (vii), which is predicted to introduce a translational stop codon, thereby leading to loss of tumor-suppressor function. APOBEC-1 overexpression also leads to promiscuous editing of multiple cytidine bases in NAT1, introducing multiple stop codons (viii), which again results in the loss of protein expression.
editing of apoB has important consequences for mammalian lipoprotein metabolism [5]. RNA editing: biochemical mechanisms A ! I editing is mediated by members of a gene family that work alone [1,6]. Adenosine deaminases that act on RNA (ADARs) contain a variable N-terminal region, followed by two or three double-stranded-RNA-binding domains and a catalytic deaminase domain [1,6]. There are three known ADARs in vertebrates: ADAR1 and ADAR2 are widely expressed in mammals, consistent with the presence of inosine-containing mRNAs in multiple tissues [7], but ADAR3 is restricted to the brain, where it binds to both single- and double-stranded mRNA [8,9]. No RNA target for ADAR3 has yet been demonstrated, although it http://tmm.trends.com
competitively inhibits the enzymatic activity of the other two ADARs [8,9]. Little is known about the role of other proteins that participate in modulating the activity of ADARs. C ! U RNA editing is mediated by a multicomponent holoenzyme complex that contains a core enzyme composed of apoB-mRNA-editing-enzyme catalytic polypeptide 1 (APOBEC-1) (an RNA-specific cytidine deaminase) and APOBEC-1 complementation factor (ACF) (an RNArecognition subunit) [10 – 12]. The target-sequence specificity of RNA editing is tightly controlled by cis-acting sequence requirements that include an AU-rich context and sequence elements flanking the targeted base [13,14], together with an optimal stoichiometry and distribution of APOBEC-1 and ACF. APOBEC-1 is developmentally
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regulated, and is expressed exclusively in the luminal gastrointestinal tract of humans [15,16]. ACF has a wide tissue distribution in both humans and other mammals, and functions as both an APOBEC-1-binding protein and an apoB-mRNA-binding protein (Fig. 1a) [10,11]. Both proteins are required for RNA editing, and regulated nuclear–cytoplasmic distribution might have a key role in determining the C ! U editing activity of the holoenzyme [17,18]. Other proteins also participate in forming the holoenzyme complex, some of which bind apoB RNA and/or APOBEC-1 (Fig. 1a) [19 – 25]. Alterations in the levels of these auxiliary proteins, which could in turn alter the availability and/or distribution of APOBEC-1 and/or ACF, might represent a mechanism by which C ! U RNAediting activity is regulated (Fig. 1a). C ! U RNA editing and cancer Dysregulated expression of APOBEC-1 is associated with aberrant RNA editing of nuclear targets A cancer phenotype emerged in the course of studies examining the effects of hepatic overexpression of APOBEC-1 in transgenic mice and rabbits [26]. Surprisingly, many animals with high-copy-number transgene expression developed hepatic dysplasia and hepatocellular carcinoma [26]. The transgenic animals lost the sitespecificity of C ! U editing of apoB RNA, normally one base in , 14 000, and instead multiple cytidines in hepatic mRNAs underwent deamination [27]. A search for candidate tumor-suppressor genes in which aberrant C ! U RNA editing had occurred led to the identification of NAT1, encoding a novel translation inhibitor, which was extensively edited by C ! U modifications (Fig. 1b). These alterations produced multiple termination codons, which greatly reduced levels of NAT1 protein within hepatocytes of APOBEC-1-transgenic animals [28]. NAT1 is homologous to the carboxyl-terminal two-thirds of eukaryotic translation-initiation-factor 4G (eIF4G), and binds to eIF4A to inhibit mRNA translation [28– 33]. Further studies demonstrated that NAT1 disruption in embryonic stem cells results in selective impairment of developmentally regulated, retinoic-acid-responsive genes, including that encoding p21WAF1, along with failure of gastrulation and defective embryogenesis [34]. These findings suggest that non-canonical C ! U RNA editing might have a role in disrupting NAT1 expression, with downstream effects on a retinoic-acid-sensitive pathway required for cellular differentiation and embryonic development [34]. The mechanisms by which alterations in NAT1 expression lead to dysplasia and cancer in adult tissues remain unknown, but it might be possible to use microarray analysis of these tumors to determine the pattern of genetic alterations that accompany this phenotype. Additional confirmation of these findings, perhaps using alternative, tissue-specific promoters would also be a welcome advance. Lower levels of transgenic APOBEC-1 expression on a wild-type background did not lead to the promiscuous editing of cytidines, suggesting that the abundance of APOBEC-1 might be a determining factor in restricting cytidine-deaminase activity to a single site in apoB RNA [35]. However, recent findings offer an alternative http://tmm.trends.com
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explanation of the role of APOBEC-1 in promiscuous RNA editing. Using a tetracycline-regulated transgene, Hersberger and colleagues introduced APOBEC-1 into the liver of APOBEC-1 2/2 mice, and examined editing of both apoB and NAT1. They found that the conditional reintroduction of even modest amounts of APOBEC-1 into the liver of APOBEC-1 2/2 mice resulted in editing of NAT1 [36]. Furthermore, this editing of NAT1 mRNA occurred in the setting of apoB RNA editing at levels below those encountered under physiological conditions in wildtype mice. Thus, introduction of APOBEC-1, at levels sufficient to induce only low levels of apoB mRNA editing, resulted in promiscuous editing of other targets. These findings suggest that supplementation of available cytoplasmic APOBEC-1, presumably beyond what can be physiologically sequestered by ACF and other auxiliary factors, might lead to indiscriminate C ! U editing and promote instability at the level of the transcriptome. This hypothesis is illustrated in Fig. 1b. A second example of aberrant C ! U RNA editing associated with a malignant phenotype involves the mRNA encoding the neurofibromatosis type 1 (NF1) protein. In this case, the CGA codon at position 3916 is altered to UGA in the edited NF1 transcript, a change that is predicted to result in translational termination and inactivation of the neurofibromin tumor-suppressor function, probably by eliminating the GTPase-activating domain encoded downstream of the edited base [37]. Skuse and colleagues first demonstrated C ! U RNA editing of NF1 in a subset of peripheral-nerve-sheath tumors [38], and noted that increased levels of C ! U editing of NF1 were also associated with a more malignant tumor phenotype [39]. A group of 34 tumors was recently examined with a view to understanding the heterogeneity of RNA editing [40], and two distinguishing features were observed in tumors that demonstrated C ! U editing of NF1. First, these tumors showed increased levels of an alternatively spliced exon 23A downstream of the edited base [40], and second, they expressed APOBEC-1 mRNA, which is normally expressed only in epithelial cells of the intestinal tract [40]. The working hypothesis for this gainof-function effect is that alternative splicing of NF1 mRNA, leading to the inclusion of exon 23A, yields a nuclear transcript configuration that permits site-specific (but non-canonical) C ! U RNA editing in the presence of aberrant APOBEC-1 expression (Fig. 1b). This introduces a translational stop codon into the edited mRNA, but it remains to be demonstrated whether the edited NF1 mRNA is translated as a truncated protein or is subject to nonsense-mediated mRNA decay. Dysregulated expression of APOBEC-1 and auxiliary proteins is associated with changes in cytoplasmic targets APOBEC-1 is an AU-rich RNA-binding protein, but lacks the typical signature RNA-binding motifs [41,42]. Several groups have demonstrated that APOBEC-1 binds apoB mRNA near the edited base and that this region, which is enriched in A and U residues, probably adopts a secondary structure that allows it to fit into the active site of the holoenzyme [43,44]. Circular-permutation analysis has
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revealed two APOBEC-1-binding sites, a major site (UUUGAU) and a minor site (UU), both located 30 of the edited base [42]. NF1 RNA also contains an APOBEC-1 consensus binding site (UUUAUU), located immediately upstream of the edited site at position 3916, and recombinant APOBEC-1 has been shown to bind NF1 RNA [40]. Independent of its role in C ! U RNA editing, there is evidence for a role of APOBEC-1 in binding to and stabilizing RNA targets that contain AU-rich motifs [42]. AU-rich sequence elements (AREs) are found in the 30 untranslated region (30 UTR) of many proto-oncogene and cytokine mRNAs, and act as an mRNA instability determinants or as a translation inhibitory elements [45]. The high-affinity APOBEC-1-binding site is contained within the minimal nine-nucleotide instability element [UUAUUU(A/U)(A/U)] previously mapped by mutational analyses [46], and further study has revealed that APOBEC-1 binds with high affinity to AREs in the 30 UTR of several transcripts, including the mRNAs encoding interleukin-12 and c-myc [42]. Furthermore, conditional overexpression of APOBEC-1 in F442A pre-adipocyte cells led to the stabilization of c-myc RNA, an effect that was dependent on crucial residues that mediate the RNA-binding function of APOBEC-1, independent of its cytidine-deaminase activity [42]. These findings suggest that altered (either increased or decreased) expression of APOBEC-1 might in turn alter the stability or metabolism of rapidly degraded RNAs, some of which are involved in regulating cell growth and proliferation in the setting of carcinogenesis. Recent findings suggest that APOBEC-1 might regulate intestinal stem-cell survival through alterations in the stability of the mRNA encoding cyclooxygenase-2 (COX-2), an additional mRNA with an ARE containing the canonical APOBEC-1-binding site [47]. Another recent development is the finding that CUGBP2 (CUG-triplet-repeat RNA-binding protein 2), an apoB-RNAediting-holoenzyme-associated protein, undergoes rapid translocation from the nucleus to the cytoplasm following radiation injury, and binds with high affinity to a 60nucleotide region in the 30 UTR of COX-2 mRNA [48]. This leads to stabilization of the mRNA but paradoxically inhibits translation of the protein [48]. COX-2 is a dominant genetic restriction point in prostaglandin biosynthesis, particularly in the intestinal epithelium, and alterations in COX-2 expression are highly correlated with colonic polyposis syndromes and with adenocarcinoma susceptibility [49–51]. Events that lead to raised COX-2 mRNA levels in the intestinal epithelium are associated with increased tumorigenesis in both experimental animals and in humans, whereas genetic or pharmacological strategies that reduce COX-2 expression are protective [52–56]. These independent demonstrations are consistent with the underlying hypothesis that aberrant or dysregulated expression of apoB-RNA-editing-associated proteins might in turn modulate the expression of cytoplasmic RNAs with demonstrated relevance to malignant transformation. Altered expression of C ! U RNA-editing proteins in malignancy Studies of APOBEC-1 expression in a variety of human cancers have produced some important results [57,58]. http://tmm.trends.com
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Elevated levels of APOBEC-1 mRNA have been observed in several human gastric, pancreatic and colonic tumors, and also in some hepatic metastases [57,58]. However, of greater significance is the striking increase in the expression of an alternatively spliced form of APOBEC-1 mRNA in which a frame shift is introduced [57,58]. This form of APOBEC-1 mRNA generates a transcript encoding a peptide made up of 36 amino acids, which shares only the N-terminal six residues with APOBEC-1 and has no enzymatic activity [57,58]. The net effect on the level of APOBEC-1 protein remains unknown, because the protein abundance is generally too low to be revealed by conventional detection methods. However, the shift in the splicing of APOBEC-1 mRNA, favoring translation of the truncated peptide, would be expected to reduce expression of the full-length protein. To better understand the regulation of APOBEC-1 expression during colorectal cancer development, rats were treated with the carcinogen, azoxymethane, and colonic epithelium examined at stages of adenoma throughout carcinoma formation [59]. A shift in promoter use in the rat APOBEC-1 gene was observed, favoring production of an mRNA with a 50 UTR that contained multiple in-frame AUG codons. This pattern of transcription was associated with decreased translation and an , 90% reduction in the abundance of APOBEC-1 protein [59]. These findings, together with evidence from human gastrointestinal tract tumors, suggest that APOBEC-1 expression might be decreased in some tumors, either through alternative splicing and production of a truncated peptide (human), or via the use of an alternative promoter and decreased translation from an AUG-burdened leader sequence (rat). A role for APOBEC-1 and related proteins in DNA mutagenesis? APOBEC-1 is member of a family of cytidine deaminases that act on monomeric nucleosides and nucleotides. However, recent findings have demonstrated that an APOBEC-1 homolog, activation-induced deaminase (AID), induces nucleotide transitions at dC and dG when expressed in Escherichia coli, in a manner that is augmented in the absence of uracil-DNA glycosylase [60]. This work suggests a mechanism of action consistent with deamination of dC residues at specific locations within DNA. Furthermore, Harris et al. have shown that APOBEC-1 itself has DNA-mutagenic activity in this assay, as do two other homologs APOBEC3C and APOBEC3G, in a manner that is consistent with dC deamination of DNA [61]. Interestingly, the three deaminases studied demonstrate striking differences in their sequence context preference of dC residues targeted for mutation. Several homologs of APOBEC-1 are expressed in human tissues, as well as in a variety of tumors and cancer cell lines [62]. In particular, the mRNA abundance for three homologs (APOBEC3B, APOBEC3C and APOBEC3G) was found to be increased in colorectal carcinoma [62]. Several homologs of APOBEC-1 can dimerize with APOBEC-1 and, thus, could potentially function as inhibitors of C ! U editing activity in trans [63,64].
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Indeed, one homolog, APOBEC2 (also known as ARCD1), has been shown to inhibit the apoB-RNA-editing activity of APOBEC-1 in cultured cells [64]. Whether these interactions could inhibit dC deamination of DNA remains to be demonstrated. Nevertheless, these data suggest that alterations in the expression of APOBEC-1 or of homologous family members, might result in cytidine deamination and mutagenesis of targets beyond the canonical site in apoB RNA. Altered expression of A ! I RNA-editing proteins in malignancy One of the most extensively examined substrates for A ! I RNA editing is the transcript encoding the brain-specific glutamate receptor subunit GluR-B. Two major sites of amino acid modification have been demonstrated. These include the Q/R site, which is edited to virtually 100% under physiological conditions [65,66]. Patients with malignant glioblastomas demonstrated reduced mRNA editing at the Q/R site, to ,70% of control levels [4]. Similar reductions in GluR-B editing in mice resulted in premature death, associated with epileptic seizures [67,68]; because epileptic seizures are an important clinical manifestation of glioblastomas, it is possible that one contributing factor might have been decreased editing of the mRNA encoding the GluR-B receptor [4]. Decreased A ! I RNA editing of the GluR-B receptor was also demonstrated at a second site (R/G) in these tumors, suggesting that the effects are probably mediated through alterations in enzymatic activity rather than changes in the target transcript structure or distribution [4]. The changes in A ! I RNAediting activity were not accompanied by changes in the abundance of the mRNA encoding either of the ADAR enzymes known to be involved (ADAR1 and ADAR2), raising the possibility that alterations in the protein distribution or the presence of a dominant inhibitor might account for the changes in editing activity [4]. Concluding remarks and outstanding questions RNA editing is an important mechanism by which a finite genome can be selectively modified and by which conditional plasticity can be generated at the level of the transcriptome. An unfortunate side effect of this plasticity is that aberrant expression of the machinery might lead to unanticipated nucleoside and nucleotide modifications, either as a cause or as a consequence of genetic instability in cancer. Important topics for future study include understanding the role of RNA-editing proteins in the selective mutagenesis of DNA, as well as in posttranscriptional mRNA processing. It will be informative to examine the cellular distribution of these proteins in relation to RNA splicing and editing, and how their partitioning within the nucleus is regulated during growth, development and aging. Finally, a major challenge is to understand the range of potential targets for such proteins, particularly those related to APOBEC-1, which appear to have arisen from a common ancestral gene. Acknowledgements We thank Valerie Blanc, Jeffrey Henderson and Debnath Mukhopadhyay for helpful discussions in the course of preparing this article. Work cited http://tmm.trends.com
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from our laboratories is supported by grants from the National Institutes of Health (HL-62265, DK-52574, HL-38180 and DK-56260). S.A. is a Research Scholar of American Gastroenterological Association.
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within AU-rich elements may be UUAUUUA(U/A)(U/A). Mol. Cell. Biol. 14, 7984 – 7995 Anant, S. et al. (2002) Apobec-1 regulates intestinal stem cell survival after radiation injury in association with posttranscriptional effects on COX-2 mRNA stability. Gastroenterology 122, A95 Mukhopadhyay, D. et al. (2003) Coupled mRNA stabilization and translational silencing of cyclooxygenase-2 by a novel RNA binding protein, CUGBP2. Mol. Cell 11, 113 – 126 Williams, C.S. et al. (1999) The role of cyclooxygenases in inflammation, cancer, and development. Oncogene 18, 7908 – 7916 Williams, C.S. and DuBois, R.N. (1996) Prostaglandin endoperoxide synthase: why two isoforms? Am. J. Physiol. 3, G393– G400 Shao, J. et al. (2000) Regulation of constitutive cyclooxygenase-2 expression in colon carcinoma cells. J. Biol. Chem. 275, 33951 – 33956 Turini, M.E. and DuBois, R.N. (2002) Cyclooxygenase-2: a therapeutic target. Annu. Rev. Med. 53, 35 – 57 Sheng, G.G. et al. (1997) A selective cyclooxygenase 2 inhibitor suppresses the growth of H-ras-transformed rat intestinal epithelial cells. Gastroenterology 113, 1883 – 1891 Giardiello, F.M. et al. (1998) Prostaglandin levels in human colorectal mucosa: effects of sulindac in patients with familial adenomatous polyposis. Dig. Dis. Sci. 43, 311 – 316 Marnett, L.J. and DuBois, R.N. (2002) COX-2: a target for colon cancer prevention. Annu. Rev. Pharmacol. Toxicol. 42, 55 – 80 Pyo, H. et al. (2001) A selective cyclooxygenase-2 inhibitor NS-398, enhances the effect of radiation in vitro and in vivo preferentially on the cells that express cyclooxygenase-2. Clin. Cancer Res. 7, 2998– 3005 Lee, R.M. et al. (1998) An alternatively spliced form of apobec-1 messenger RNA is overexpressed in human colon cancer. Gastroenterology 115, 1096– 1103 Greeve, J. et al. (1999) Absence of APOBEC-1 mediated mRNA editing in human carcinomas. Oncogene 18, 6357– 6366 Anant, S. et al. (2002) Apobec-1 transcription in rat colon cancer: decreased apobec-1 protein production through alterations in polysome distribution and mRNA translation associated with upstream AUGs. Biochim. Biophys. Acta 1575, 54 – 62 Petersen-Mahrt, S.K. et al. (2002) AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418, 99 – 103 Harris, R.S. et al. (2002) RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators. Mol. Cell 10, 1247– 1253 Jarmuz, A. et al. (2002) An anthropoid-specific locus of orphan C to U RNA-editing enzymes on chromosome 22. Genomics 79, 285– 296 Liao, W. et al. (1999) APOBEC-2, a cardiac- and skeletal musclespecific member of the cytidine deaminase supergene family. Biochem. Biophys. Res. Commun. 260, 398– 404 Anant, S. et al. (2001) ARCD-1, an apobec-1-related cytidine deaminase, exerts a dominant negative effect on C to U RNA editing. Am. J. Physiol. Cell Physiol. 281, C1904 – C1916 Sommer, B. et al. (1991) RNA editing in brain controls a determinant of ion flow in glutamate-gated channels. Cell 67, 11 – 19 Paschen, W. et al. (1994) RNA editing of the glutamate receptor subunits GluR2 and GluR6 in human brain tissue. J. Neurochem. 63, 1596– 1602 Brusa, R. et al. (1995) Early-onset epilepsy and postnatal lethality associated with an editing-deficient GluR-B allele in mice. Science 270, 1677– 1680 Higuchi, M. et al. (2000) Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2. Nature 406, 78 – 81
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Hydrolytic nucleoside and nucleotide deamination, and genetic instability: a possible link between RNA-editing enzymes and cancer? Shrikant Anant1 and Nicholas O. Davidson2 1
Department of Internal Medicine, Washington University School of Medicine, Saint Louis, MO 63110, USA Departments of Internal Medicine, and Molecular Biology and Pharmacology, Washington University School of Medicine, Saint Louis, MO 63110, USA 2
Post-transcriptional RNA editing generates novel gene products by changing the coding sequence of the transcript from that in the genome. Two classes of RNA editing exist in mammals, each of which involves an enzymatic deamination. These reactions have stringent sequence and structural requirements for their target RNAs, and each requires distinctive enzymatic machinery. Alterations in the expression or abundance of RNAediting factors produce unanticipated alterations in the processing or expression of RNAs, in some cases outside their physiological targets. Recent findings suggest that unregulated expression of the cytidine-deaminase gene family might lead to deamination of deoxycytidine nucleotides in DNA. Aberrant or dysregulated RNA editing, or altered expression of editing factors, might contribute to genomic instability in cancer. Several mechanisms exist by which organisms can expand their genetic repertoire without creating a new gene. These include the use of alternative promoters to create different mRNA transcripts from the same gene, and a variety of intranuclear, post-transcriptional processing and modification steps, such as splicing, editing and polyadenylation, by which the sequence of the primary transcript can be altered from that specified in the chromosomal gene. These various adaptations create a hierarchy of alternative coding templates or differentially processed transcripts, and thus permit considerable plasticity within the context of a single gene. In this article, we discuss the evidence that dysregulation of a specific class of post-transcriptional modification, RNA editing, might be associated with cancer. The underlying hypothesis is that aberrant RNA editing, mediated through alterations in the expression of one or more trans-acting factors, can lead to unanticipated alterations in the sequence and/or function of RNA transcripts beyond their canonical target. Recent findings also suggest that uncontrolled expression of protein components of the RNA-editing enzyme complexes might result in Corresponding author: Nicholas O. Davidson ([email protected]).
promiscuous nucleotide deamination in DNA. These aberrant processing events occur in human cancers and in experimental animal models, and emphasize the need to consider this type of gene regulation in the wider context of factors that contribute to genetic instability. RNA editing: overview and physiological regulation Post-transcriptional RNA editing is a process by which the nucleotide sequence of a nuclear transcript is changed from that encoded in the genomic template. This can alter the codon reading frame and amino acid sequence of the translation product, thereby changing the function of the gene product. In mammals, RNA editing occurs through base modification, via deamination of cytidine (C) to uridine (U) or of adenosine (A) to inosine (I), within particular nuclear mRNAs. A ! I RNA editing occurs on unspliced RNA templates and requires base-pairing between an exon and an adjacent intron in the targeted RNA [1 –3]. Inosine is recognized by the translational apparatus as guanosine, so the net effect of A ! I RNA editing is changes an A to a G in the reading frame, which in turn alters the sequence and function of the gene product [1 – 3]. Examples of mRNAs in which A ! I RNA editing occurs include those encoding the calcium-gated glutamate receptor GluR-B, hepatitis delta virus and the 5-hydroxytryptamine (5-HT2c) receptor [4]. The original and best-characterized example of C ! U editing is the mRNA encoding apolipoprotein B (apoB), in which a single, site-specific cytidine deamination introduces a translational termination codon (UAA) into the reading frame of the edited transcript, leading to production of the shortened isoform, apoB48 (Fig. 1a). This occurs exclusively in the human small intestine, where apoB48 is required for the absorption of dietary lipid [5]. ApoB RNA expressed in human liver is not edited and hepatic apoB RNA encodes a larger protein, apoB100. The carboxyl terminus of apoB100, which contains the binding domain for the low-density-lipoprotein receptor, is absent from the intestinal form (apoB48). Hence, C ! U RNA
http://tmm.trends.com 1471-4914/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1471-4914(03)00032-7
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Holoenzyme complex for apoB RNA editing TRENDS in Molecular Medicine
Fig. 1. Site-specific deamination of cytidine (C) to uridine (U) (C ! U RNA editing) and the consequences of aberrant expression of the gene encoding apolipoprotein-BmRNA-editing-enzyme catalytic polypeptide 1 (APOBEC-1). (a) Under physiological conditions, C ! U RNA editing targets a single cytidine nucleotide in the nuclear transcript encoding intestinal apolipoprotein B (apoB). The C ! U editing enzyme complex includes a catalytic subunit APOBEC-1 (green), and an RNA-recognition subunit APOBEC-1 complementation factor (ACF) (pink). ACF binds APOBEC-1 and forms a higher-order complex within the nucleus. Several constraints could potentially act as regulatory points in this process. ACF is predominantly nuclear and its partitioning is distinct from that of APOBEC-1, which is predominantly cytoplasmic (i). APOBEC-1 binds to ACF as well as other auxiliary proteins [such as GRY-RBP (glycine-, arginine- and tyrosine-rich RNA-binding protein) and CUGBP2 (CUG-triplet-repeat RNA-binding protein 2)] (blue) in the cytoplasm (ii), and in the nucleus (iii). C ! U RNA editing of apoB requires an optimal nuclear stoichiometry of ACF and APOBEC-1 within the context of a holoenzyme (iv), which allows targeted cytidine deamination of a single base in nuclear apoB mRNA, resulting in the production of apoB48. (b) Aberrant regulation of APOBEC-1 expression leads to unanticipated changes in transcripts other than apoB. If APOBEC-1 is overexpressed (either forced transgenic or sporadic overexpression) (i), cytoplasmic transcripts containing AU-rich elements might be bound by APOBEC-1 (ii), causing alterations in their stability and/or translation (iii). In addition, increased abundance of APOBEC-1 could result in increased diffusion or transport into the nucleus (iv) where other mRNA targets might be susceptible to enzymatic deamination. These include the mRNAs encoding neurofibromatosis type 1 (NF1) (v) and NAT1 (vi). APOBEC-1 overexpression results in C ! U editing at a single site in NF1 (vii), which is predicted to introduce a translational stop codon, thereby leading to loss of tumor-suppressor function. APOBEC-1 overexpression also leads to promiscuous editing of multiple cytidine bases in NAT1, introducing multiple stop codons (viii), which again results in the loss of protein expression.
editing of apoB has important consequences for mammalian lipoprotein metabolism [5]. RNA editing: biochemical mechanisms A ! I editing is mediated by members of a gene family that work alone [1,6]. Adenosine deaminases that act on RNA (ADARs) contain a variable N-terminal region, followed by two or three double-stranded-RNA-binding domains and a catalytic deaminase domain [1,6]. There are three known ADARs in vertebrates: ADAR1 and ADAR2 are widely expressed in mammals, consistent with the presence of inosine-containing mRNAs in multiple tissues [7], but ADAR3 is restricted to the brain, where it binds to both single- and double-stranded mRNA [8,9]. No RNA target for ADAR3 has yet been demonstrated, although it http://tmm.trends.com
competitively inhibits the enzymatic activity of the other two ADARs [8,9]. Little is known about the role of other proteins that participate in modulating the activity of ADARs. C ! U RNA editing is mediated by a multicomponent holoenzyme complex that contains a core enzyme composed of apoB-mRNA-editing-enzyme catalytic polypeptide 1 (APOBEC-1) (an RNA-specific cytidine deaminase) and APOBEC-1 complementation factor (ACF) (an RNArecognition subunit) [10 – 12]. The target-sequence specificity of RNA editing is tightly controlled by cis-acting sequence requirements that include an AU-rich context and sequence elements flanking the targeted base [13,14], together with an optimal stoichiometry and distribution of APOBEC-1 and ACF. APOBEC-1 is developmentally
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regulated, and is expressed exclusively in the luminal gastrointestinal tract of humans [15,16]. ACF has a wide tissue distribution in both humans and other mammals, and functions as both an APOBEC-1-binding protein and an apoB-mRNA-binding protein (Fig. 1a) [10,11]. Both proteins are required for RNA editing, and regulated nuclear–cytoplasmic distribution might have a key role in determining the C ! U editing activity of the holoenzyme [17,18]. Other proteins also participate in forming the holoenzyme complex, some of which bind apoB RNA and/or APOBEC-1 (Fig. 1a) [19 – 25]. Alterations in the levels of these auxiliary proteins, which could in turn alter the availability and/or distribution of APOBEC-1 and/or ACF, might represent a mechanism by which C ! U RNAediting activity is regulated (Fig. 1a). C ! U RNA editing and cancer Dysregulated expression of APOBEC-1 is associated with aberrant RNA editing of nuclear targets A cancer phenotype emerged in the course of studies examining the effects of hepatic overexpression of APOBEC-1 in transgenic mice and rabbits [26]. Surprisingly, many animals with high-copy-number transgene expression developed hepatic dysplasia and hepatocellular carcinoma [26]. The transgenic animals lost the sitespecificity of C ! U editing of apoB RNA, normally one base in , 14 000, and instead multiple cytidines in hepatic mRNAs underwent deamination [27]. A search for candidate tumor-suppressor genes in which aberrant C ! U RNA editing had occurred led to the identification of NAT1, encoding a novel translation inhibitor, which was extensively edited by C ! U modifications (Fig. 1b). These alterations produced multiple termination codons, which greatly reduced levels of NAT1 protein within hepatocytes of APOBEC-1-transgenic animals [28]. NAT1 is homologous to the carboxyl-terminal two-thirds of eukaryotic translation-initiation-factor 4G (eIF4G), and binds to eIF4A to inhibit mRNA translation [28– 33]. Further studies demonstrated that NAT1 disruption in embryonic stem cells results in selective impairment of developmentally regulated, retinoic-acid-responsive genes, including that encoding p21WAF1, along with failure of gastrulation and defective embryogenesis [34]. These findings suggest that non-canonical C ! U RNA editing might have a role in disrupting NAT1 expression, with downstream effects on a retinoic-acid-sensitive pathway required for cellular differentiation and embryonic development [34]. The mechanisms by which alterations in NAT1 expression lead to dysplasia and cancer in adult tissues remain unknown, but it might be possible to use microarray analysis of these tumors to determine the pattern of genetic alterations that accompany this phenotype. Additional confirmation of these findings, perhaps using alternative, tissue-specific promoters would also be a welcome advance. Lower levels of transgenic APOBEC-1 expression on a wild-type background did not lead to the promiscuous editing of cytidines, suggesting that the abundance of APOBEC-1 might be a determining factor in restricting cytidine-deaminase activity to a single site in apoB RNA [35]. However, recent findings offer an alternative http://tmm.trends.com
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explanation of the role of APOBEC-1 in promiscuous RNA editing. Using a tetracycline-regulated transgene, Hersberger and colleagues introduced APOBEC-1 into the liver of APOBEC-1 2/2 mice, and examined editing of both apoB and NAT1. They found that the conditional reintroduction of even modest amounts of APOBEC-1 into the liver of APOBEC-1 2/2 mice resulted in editing of NAT1 [36]. Furthermore, this editing of NAT1 mRNA occurred in the setting of apoB RNA editing at levels below those encountered under physiological conditions in wildtype mice. Thus, introduction of APOBEC-1, at levels sufficient to induce only low levels of apoB mRNA editing, resulted in promiscuous editing of other targets. These findings suggest that supplementation of available cytoplasmic APOBEC-1, presumably beyond what can be physiologically sequestered by ACF and other auxiliary factors, might lead to indiscriminate C ! U editing and promote instability at the level of the transcriptome. This hypothesis is illustrated in Fig. 1b. A second example of aberrant C ! U RNA editing associated with a malignant phenotype involves the mRNA encoding the neurofibromatosis type 1 (NF1) protein. In this case, the CGA codon at position 3916 is altered to UGA in the edited NF1 transcript, a change that is predicted to result in translational termination and inactivation of the neurofibromin tumor-suppressor function, probably by eliminating the GTPase-activating domain encoded downstream of the edited base [37]. Skuse and colleagues first demonstrated C ! U RNA editing of NF1 in a subset of peripheral-nerve-sheath tumors [38], and noted that increased levels of C ! U editing of NF1 were also associated with a more malignant tumor phenotype [39]. A group of 34 tumors was recently examined with a view to understanding the heterogeneity of RNA editing [40], and two distinguishing features were observed in tumors that demonstrated C ! U editing of NF1. First, these tumors showed increased levels of an alternatively spliced exon 23A downstream of the edited base [40], and second, they expressed APOBEC-1 mRNA, which is normally expressed only in epithelial cells of the intestinal tract [40]. The working hypothesis for this gainof-function effect is that alternative splicing of NF1 mRNA, leading to the inclusion of exon 23A, yields a nuclear transcript configuration that permits site-specific (but non-canonical) C ! U RNA editing in the presence of aberrant APOBEC-1 expression (Fig. 1b). This introduces a translational stop codon into the edited mRNA, but it remains to be demonstrated whether the edited NF1 mRNA is translated as a truncated protein or is subject to nonsense-mediated mRNA decay. Dysregulated expression of APOBEC-1 and auxiliary proteins is associated with changes in cytoplasmic targets APOBEC-1 is an AU-rich RNA-binding protein, but lacks the typical signature RNA-binding motifs [41,42]. Several groups have demonstrated that APOBEC-1 binds apoB mRNA near the edited base and that this region, which is enriched in A and U residues, probably adopts a secondary structure that allows it to fit into the active site of the holoenzyme [43,44]. Circular-permutation analysis has
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revealed two APOBEC-1-binding sites, a major site (UUUGAU) and a minor site (UU), both located 30 of the edited base [42]. NF1 RNA also contains an APOBEC-1 consensus binding site (UUUAUU), located immediately upstream of the edited site at position 3916, and recombinant APOBEC-1 has been shown to bind NF1 RNA [40]. Independent of its role in C ! U RNA editing, there is evidence for a role of APOBEC-1 in binding to and stabilizing RNA targets that contain AU-rich motifs [42]. AU-rich sequence elements (AREs) are found in the 30 untranslated region (30 UTR) of many proto-oncogene and cytokine mRNAs, and act as an mRNA instability determinants or as a translation inhibitory elements [45]. The high-affinity APOBEC-1-binding site is contained within the minimal nine-nucleotide instability element [UUAUUU(A/U)(A/U)] previously mapped by mutational analyses [46], and further study has revealed that APOBEC-1 binds with high affinity to AREs in the 30 UTR of several transcripts, including the mRNAs encoding interleukin-12 and c-myc [42]. Furthermore, conditional overexpression of APOBEC-1 in F442A pre-adipocyte cells led to the stabilization of c-myc RNA, an effect that was dependent on crucial residues that mediate the RNA-binding function of APOBEC-1, independent of its cytidine-deaminase activity [42]. These findings suggest that altered (either increased or decreased) expression of APOBEC-1 might in turn alter the stability or metabolism of rapidly degraded RNAs, some of which are involved in regulating cell growth and proliferation in the setting of carcinogenesis. Recent findings suggest that APOBEC-1 might regulate intestinal stem-cell survival through alterations in the stability of the mRNA encoding cyclooxygenase-2 (COX-2), an additional mRNA with an ARE containing the canonical APOBEC-1-binding site [47]. Another recent development is the finding that CUGBP2 (CUG-triplet-repeat RNA-binding protein 2), an apoB-RNAediting-holoenzyme-associated protein, undergoes rapid translocation from the nucleus to the cytoplasm following radiation injury, and binds with high affinity to a 60nucleotide region in the 30 UTR of COX-2 mRNA [48]. This leads to stabilization of the mRNA but paradoxically inhibits translation of the protein [48]. COX-2 is a dominant genetic restriction point in prostaglandin biosynthesis, particularly in the intestinal epithelium, and alterations in COX-2 expression are highly correlated with colonic polyposis syndromes and with adenocarcinoma susceptibility [49–51]. Events that lead to raised COX-2 mRNA levels in the intestinal epithelium are associated with increased tumorigenesis in both experimental animals and in humans, whereas genetic or pharmacological strategies that reduce COX-2 expression are protective [52–56]. These independent demonstrations are consistent with the underlying hypothesis that aberrant or dysregulated expression of apoB-RNA-editing-associated proteins might in turn modulate the expression of cytoplasmic RNAs with demonstrated relevance to malignant transformation. Altered expression of C ! U RNA-editing proteins in malignancy Studies of APOBEC-1 expression in a variety of human cancers have produced some important results [57,58]. http://tmm.trends.com
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Elevated levels of APOBEC-1 mRNA have been observed in several human gastric, pancreatic and colonic tumors, and also in some hepatic metastases [57,58]. However, of greater significance is the striking increase in the expression of an alternatively spliced form of APOBEC-1 mRNA in which a frame shift is introduced [57,58]. This form of APOBEC-1 mRNA generates a transcript encoding a peptide made up of 36 amino acids, which shares only the N-terminal six residues with APOBEC-1 and has no enzymatic activity [57,58]. The net effect on the level of APOBEC-1 protein remains unknown, because the protein abundance is generally too low to be revealed by conventional detection methods. However, the shift in the splicing of APOBEC-1 mRNA, favoring translation of the truncated peptide, would be expected to reduce expression of the full-length protein. To better understand the regulation of APOBEC-1 expression during colorectal cancer development, rats were treated with the carcinogen, azoxymethane, and colonic epithelium examined at stages of adenoma throughout carcinoma formation [59]. A shift in promoter use in the rat APOBEC-1 gene was observed, favoring production of an mRNA with a 50 UTR that contained multiple in-frame AUG codons. This pattern of transcription was associated with decreased translation and an , 90% reduction in the abundance of APOBEC-1 protein [59]. These findings, together with evidence from human gastrointestinal tract tumors, suggest that APOBEC-1 expression might be decreased in some tumors, either through alternative splicing and production of a truncated peptide (human), or via the use of an alternative promoter and decreased translation from an AUG-burdened leader sequence (rat). A role for APOBEC-1 and related proteins in DNA mutagenesis? APOBEC-1 is member of a family of cytidine deaminases that act on monomeric nucleosides and nucleotides. However, recent findings have demonstrated that an APOBEC-1 homolog, activation-induced deaminase (AID), induces nucleotide transitions at dC and dG when expressed in Escherichia coli, in a manner that is augmented in the absence of uracil-DNA glycosylase [60]. This work suggests a mechanism of action consistent with deamination of dC residues at specific locations within DNA. Furthermore, Harris et al. have shown that APOBEC-1 itself has DNA-mutagenic activity in this assay, as do two other homologs APOBEC3C and APOBEC3G, in a manner that is consistent with dC deamination of DNA [61]. Interestingly, the three deaminases studied demonstrate striking differences in their sequence context preference of dC residues targeted for mutation. Several homologs of APOBEC-1 are expressed in human tissues, as well as in a variety of tumors and cancer cell lines [62]. In particular, the mRNA abundance for three homologs (APOBEC3B, APOBEC3C and APOBEC3G) was found to be increased in colorectal carcinoma [62]. Several homologs of APOBEC-1 can dimerize with APOBEC-1 and, thus, could potentially function as inhibitors of C ! U editing activity in trans [63,64].
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Indeed, one homolog, APOBEC2 (also known as ARCD1), has been shown to inhibit the apoB-RNA-editing activity of APOBEC-1 in cultured cells [64]. Whether these interactions could inhibit dC deamination of DNA remains to be demonstrated. Nevertheless, these data suggest that alterations in the expression of APOBEC-1 or of homologous family members, might result in cytidine deamination and mutagenesis of targets beyond the canonical site in apoB RNA. Altered expression of A ! I RNA-editing proteins in malignancy One of the most extensively examined substrates for A ! I RNA editing is the transcript encoding the brain-specific glutamate receptor subunit GluR-B. Two major sites of amino acid modification have been demonstrated. These include the Q/R site, which is edited to virtually 100% under physiological conditions [65,66]. Patients with malignant glioblastomas demonstrated reduced mRNA editing at the Q/R site, to ,70% of control levels [4]. Similar reductions in GluR-B editing in mice resulted in premature death, associated with epileptic seizures [67,68]; because epileptic seizures are an important clinical manifestation of glioblastomas, it is possible that one contributing factor might have been decreased editing of the mRNA encoding the GluR-B receptor [4]. Decreased A ! I RNA editing of the GluR-B receptor was also demonstrated at a second site (R/G) in these tumors, suggesting that the effects are probably mediated through alterations in enzymatic activity rather than changes in the target transcript structure or distribution [4]. The changes in A ! I RNAediting activity were not accompanied by changes in the abundance of the mRNA encoding either of the ADAR enzymes known to be involved (ADAR1 and ADAR2), raising the possibility that alterations in the protein distribution or the presence of a dominant inhibitor might account for the changes in editing activity [4]. Concluding remarks and outstanding questions RNA editing is an important mechanism by which a finite genome can be selectively modified and by which conditional plasticity can be generated at the level of the transcriptome. An unfortunate side effect of this plasticity is that aberrant expression of the machinery might lead to unanticipated nucleoside and nucleotide modifications, either as a cause or as a consequence of genetic instability in cancer. Important topics for future study include understanding the role of RNA-editing proteins in the selective mutagenesis of DNA, as well as in posttranscriptional mRNA processing. It will be informative to examine the cellular distribution of these proteins in relation to RNA splicing and editing, and how their partitioning within the nucleus is regulated during growth, development and aging. Finally, a major challenge is to understand the range of potential targets for such proteins, particularly those related to APOBEC-1, which appear to have arisen from a common ancestral gene. Acknowledgements We thank Valerie Blanc, Jeffrey Henderson and Debnath Mukhopadhyay for helpful discussions in the course of preparing this article. Work cited http://tmm.trends.com
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from our laboratories is supported by grants from the National Institutes of Health (HL-62265, DK-52574, HL-38180 and DK-56260). S.A. is a Research Scholar of American Gastroenterological Association.
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