Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2017/02/10/dmd.116.073254.DC1

1521-009X/45/4/375–389$25.00 http://dx.doi.org/10.1124/dmd.116.073254 AND DISPOSITION Drug Metab Dispos 45:375–389, April 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics Minireview

Alternative Splicing in the Superfamily Expands Diversity to Augment Function and Redirect Human Drug Metabolism s

Andrew J. Annalora, Craig B. Marcus, and Patrick L. Iversen

Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon

Received September 2, 2016; accepted February 6, 2017 Downloaded from ABSTRACT The encodes 57 cytochrome P450 , whose to a disease pathology. The expansion of P450 transcript diversity products metabolize hundreds of drugs, thousands of xeno- involves tissue-specific splicing factors, transformation-sensitive al- biotics, and unknown numbers of endogenous compounds, including ternate splicing, trans-splicing between gene transcripts, single- steroids, retinoids, and . Indeed, P450 genes are the first nucleotide polymorphisms, and epigenetic regulation of alternate line of defense against daily environmental chemical challenges in a splicing. Homeostatic regulation of variant P450 expression is influ- dmd.aspetjournals.org manner that parallels the immune system. Several National Institutes of enced also by nuclear signaling, suppression of nonsense- Health databases, including PubMed, AceView, and Ensembl, were mediated decay or premature termination codons, mitochondrial queried to establish a comprehensive analysis of the full human P450 dysfunction, or host infection. This review focuses on emergent transcriptome. This review describes a remarkable diversification of aspects of the adaptive gene-splicing process, which when viewed the 57 human P450 genes, which may be alternatively processed into through the lens of P450–nuclear receptor gene interactions, resem- nearly 1000 distinct mRNA transcripts to shape an individual’s P450 blesaprimitiveimmune-likesystemthat can rapidly monitor, respond, proteome. Important P450 splice variants from families 1A, 1B, 2C, 2D, and diversify to acclimate to fluctuations in endo-xenobiotic exposure. 3A, 4F, 19A, and 24A have now been documented, with some displaying Insights gained from this review should aid future drug discovery and at ASPET Journals on October 3, 2021 alternative subcellular distribution or catalytic function directly linked improve therapeutic management of personalized drug regimens.

Introduction transcripts and polypeptide products will improve our ability to assess the Mapping the human genome sequence was completed in 2001 (Lander functional nature of its role in both physiologic and pathologic conditions et al., 2001), helping to usher in the “postgenomic era” of personalized (La Cognata et al., 2014). The spectrum of alternate splicing mechanisms medicine. Over a decade later, however, the promise of pharmacoge- underlying the expansion of the proteome involves the use of seven main nomics (PGx) has yet to fully materialize, and the composition of the splicing types, as previously described (Blencowe, 2006; Roy et al., human genome remains enigmatic, as numerous questions linger con- 2013). The molecular basis for these mechanisms is complex and beyond cerning the complexity of its content and organization. For example, how the scope of this review; however, a brief description of the phenomenon can a genome with only 22,000 genes produce a proteome with over and its key players is provided in the supplemental materials (see 200,000 distinct ? While transcription is a relatively well un- Supplemental Fig. 1). Here we will focus primarily on the spectrum of derstood phenomenon, the mechanisms involved in regulating alternative phenotypic outcomes induced by alternative transcript splicing, in precursor or unprocessed messenger RNA (pre-mRNA) splicing, the order to highlight the array of splice-sensitive features operating in the driving force behind both transcriptome and proteome expansion, remains cytochrome P450 (P450) superfamily, a collection of 57 human genes less appreciated. The ability to link a single gene to its full suite of RNA that coordinate the metabolism of both drugs and endoxenobiotics. This work focuses new attention on the biologic impact of alternative P450 gene splicing, an underappreciated component of phase I drug metabolism that may complicate or disrupt personalized approaches to This research was supported by start-up funds from the Office of Research and medicine. Precision medicine, therefore, through the universal applica- the College of Agricultural Sciences at Oregon State University (Corvallis, OR) awarded to Drs. Marcus and Iversen. tion of genetics, still faces many challenges beyond cost, ethical dx.doi.org/10.1124/dmd.116.073254. considerations, and the need for additional, whole genome sequences. s This article has supplemental material available at dmd.aspetjournals. Multiple resources have now been developed, in addition to the org. Human Genome Project, to address these challenges, including: the

ABBREVIATIONS: BP, benzo(a)pyrene; ER, endoplasmic reticulum; GWAS, genome-wide association studies; NMD, nonsense-mediated decay; NR, nuclear receptor; P450, cytochrome P450; PGx, ; PPAR, peroxisome proliferator–activated receptors; pre-mRNA, precursor or unprocessed messenger RNA; ROS, reactive oxygen species; SNP, single-nucleotide polymorphism; snRNA, small nuclear RNA; VDR, vitamin D receptor.

375 376 Annalora et al.

Hapmap Project, which enables single-nucleotide polymorphism and intronic-splicing enhancers) with tissue-specific, trans-acting, (SNP) arrays for .100,000 SNPs; the 1000 Genomes Project; the splicing regulatory factors. In this way the same pre-mRNA transcript ENCODE project for noncoding RNAs; and the National Human can be processed into tissue-specific, alternately spliced forms (Wang Genome Research Institute (NHGRI) genome-wide association and Burge, 2008). The cytochrome P450 superfamily of genes is well studies (GWAS) catalog. GWAS have now identified two P450s studied from many perspectives (Guengerich, 2013; Johnson and that represent a biomarker for disease or a drug-response phenotype, Stout, 2013; Pikuleva and Waterman, 2013). As of late 2015, PubMed including: 1) a CYP2C8 association with bisphosphonate-related listed over 23,790 citations referencing human P450 genes (according osteonecrosis of the jaw in multiple myeloma (Sarasquete et al., to GeneCards.org), with much of this work being focused on tissue- 2008); and 2) a CYP2C19 association with interindividual specific expression or catalytic function of individual, reference forms. variation as an (Shuldiner et al., 2009). Aside from Alternative splicing of P450s has also been well studied for more than showing strong associations, few pharmacogenetic biomarkers of this two decades; however, the structural and functional diversity of P450 sort have successfully transitioned from discovery to clinical practice splice variants, and their relationship to human health and disease, (Carr et al., 2014). Many GWAS studies have failed to provide remains poorly understood. A summary of known P450 splice variants prognostic value because they are not designed to evaluate how linked to human disease is presented in Table 1. Reports from groups variant is influenced by both cellular and environ- attempting to synthesize a global view of alternative P450 splicing, mental splicing factors. In this regard, we feel a new appreciation is and its relevance to our understanding of human metabolism in the needed for the complexity of alternative splicing. For not only is the context of PGx or personalized medicine, are exceedingly rare

world of both coding and noncoding RNA more diverse and more (Nelson et al., 2004; Turman et al., 2006). Downloaded from complex than we could have imagined even a decade ago, it is teeming Our review has led us to conclude that personalized approaches to with untold amounts of RNA “dark matter” and other relics of the medicine whose sole basis is GWAS may be misleading, in that they fail RNA world, whose structure and function must be fully appreciated to account for variability in compensatory splicing mechanisms, which and classified before a new guiding orthodoxy for “individualized can modulate the expression of some highly penetrant . This medicine” can be safely conceived (St. Laurent et al., 2014; Cowie phenomenon is exemplified by ontogenetic regulatory factors that can

et al., 2015). mask the physiologic impact of genetic variations among developmen- dmd.aspetjournals.org tally regulated P450 genes (Hines and McCarver, 2002). To expand on this concept, and explore the array of alternative splicing products that Meta-Analysis of Alternative Gene Splicing in the Cytochrome serve as an improved platform for predicting disease whose basis is the P450 Superfamily functional genome, we annotated all of the known human P450 splice Alternative splicing regulated by tissue-specific factors is a means of variants currently listed in the NCBI’s PubMed, AceView, and Ensembl adapting to physiologic demands. Tissue-specific splice variants may databases (Thierry-Mieg and Thierry-Mieg, 2006; Cunningham et al., arise from tissue-specific promoter elements (Wiemann et al., 2005) or 2015) as of late 2015. From this computational meta-analysis, we found tissue-specific–splicing regulatory elements (Black, 2000). Exon and that the 57 human P450 genes produce a P450 “transcriptome cloud” at ASPET Journals on October 3, 2021 intron definition requires interaction of discrete cis-elements (exonic- composed of at least 965 unique mRNA transcripts (Table 2). This splicing enhancers, exonic-splicing silencers, intronic splicing silencers, number represents all known reference (or wild-type) and variant P450

TABLE 1 Pathologies associated with P450 splice variants

P450 Isoform Variant Form Related Disease Reference ΔExon 2 and 6 Ovarian cancer Chinta et al. (2005) CYP1A1 Bauer et al. (2007) Leung et al. (2005) Partial ΔExon 2 Glaucoma Stoilov et al. (1998) CYP1B1 Li et al. (2014) CYP2A6 Deletions Lung cancer Liu et al. (2013b) CYP2B6 ΔExons 4–6 Liver/colon cancer Miles et al. (1989) ΔExons 2 and 9 Essential hypertension Nakayama et al. (2002b) CYP8A1 Nakayama et al. (2002a) CYP11B2 Trans-splicing to CYP11B1 Congenital adrenal hyperplasia Hampf et al. (2001) ΔExons 2, 4, 6, or 8 17-a Hydroxylase deficiency, Yamaguchi et al. (1998) CYP17A1 congenital adrenal hyperplasia Costa-Santos et al. (2004) Hwang et al. (2011) CYP19A1 ΔExon 5 deficiency Pepe et al. (2007) CYP24A1 ΔExons 9–11 Breast, colon, and prostate cancer Muindi et al. (2007) ΔExon 2 Atherosclerosis Elmabsout et al. (2012) CYP26B1 Oral cancer Chen et al. (2014) ΔExons 4,6, 7 Cerebrotendinous xanthomatosis Garuti et al. (1996) CYP27A1 Verrips et al. (1997) Chen et al. (1998) ΔExons 4–7 Glioblastoma, leukemia, melanoma, Maas et al. (2001) cervical cancer, ovarian cancer Flanagan et al. (2003) CYP27B1 Fischer et al. (2007) Wu et al. (2007) Fischer et al. (2009b) Intron 2 Alzheimer disease Li et al. (2013a) CYP46A1 Li et al. (2013b) Alternative P450 Splicing Redirects Human Drug Metabolism 377 mRNAs, including some with retained introns, premature termination influence of a point on gene function. Although compu- codons (PTCs), and those subject to nonsense-mediated decay (NMD); tational studies indicate that 85% of all human mRNA comes from a we also included several experimentally derived transcripts described in single major transcript (Gonzàlez-Porta et al., 2013), the P450 PubMed-listed citations (identified by searching CYP* or P450 splice superfamily of genes is highly diverse at the genomic level, making variant or alternative CYP* or P450 splicing) that were not represented it uniquely sensitive to alternate splicing events that alter the biologic in the AceView or Ensembl databases. output of both genetic and epigenetic signaling cascades. Nuclear Our results approximate that the average P450 gene encodes nearly receptors are now recognized for their role in mediating global gene- 20 unique mRNA transcripts, capable of yielding both reference and splicing events (Auboeuf et al., 2005), and an improved framework splice-variant proteins, and an ensemble of noncoding RNA molecules. for how this process functions to regulate drug metabolism appears to Over 53% of these transcripts (515) are predicted to express viable be in order, one that can potentially recontextualize the utility and proteins, on the basis of the most stringent expression criteria used by complexity of the convoluted metabolic crosstalk network that underlies both the AceView and Ensembl databases (Table 2). Subsequent the 48 nuclear receptor (NR) signaling pathways operating in humans analysis of the GeneCards.org website revealed that the 57 human (Meyer, 2007; Tralau and Luch, 2013). P450s are associated with over 48,384 SNPs, approximating to roughly Furthermore, drug metabolizing P450s of families 1–4 have tradi- 850 mutations per P450 gene (Stelzer et al., 2011). Although most P450s tionally been considered more polymorphic than P450s that metabolize fall within this general range of genetic variation, some, including CYPs endogenous, cholesterol-based substrates and are assumed to be more 2C19, 5A1, 19A1, and 39A1, express more than twice as many SNPs sensitive to SNP-based modulation of alternative splicing (Lewiñska

as the average, suggesting the accumulation process is nonrandom et al., 2013). On the basis of our analysis, it does appear that that the Downloaded from (Table 2). The total number of diseases associated with each of the majority of steroid-metabolizing P450s, including CYP17A1 (91 SNPs) 57 human genes (1057) was also calculated using the MalaCards Human and CYP21A2 (173 SNPs), are among the least polymorphic P450 Disease Database (Rappaport et al., 2014). P450 families 1 (203) and genes. As described above, mutations in these P450 are closely 2 (413) account for over half of the total disease associations, whereas linked to congenital defects such as pseudohermaphroditism and adrenal orphan P450s CYP20A1 (1) and CYP39A1 (2) are associated with the hyperplasia (Yamaguchi et al., 1998; Doleschall et al., 2014), and SNPs

fewest. may be less well tolerated in these systems owing to their central role in dmd.aspetjournals.org A side-by-side, radar-plots comparison of the total number of the production of sex hormones, progestins, and corticoids. In contrast, alternative P450 splice variants and SNPs for each of the 57 human the 35 drug-metabolizing P450s that compose P450 families 1–4in P450 genes is shown in Fig. 1. Pharmacologically relevant drug targets, humans average over 700 SNPs per isoform, and the 25,659 total such as CYP2C9, CYP2D6, CYP3A5, CYP11A1, and CYP19A1, are polymorphisms in this group represent over 53% of the total P450 among the most highly spliced P450 genes, the majority of which are SNP population. Although CYP2C9 (1422 SNPs), CYP2C18 also subject to trans-splicing events. Although differences in gene (1230 SNPs), and CYP2C19 (2467) are the most polymorphic drug- organization or cell-specific selection pressure may play a role in this metabolizing P450s, they are considerably less polymorphic than phenomenon, our observations suggest that drug-dosing guidelines genes like CYP5A1 (5274 SNPs), which metabolizes prostaglan- at ASPET Journals on October 3, 2021 derived from knowledge of a patient’s individual P450 transcriptome dins, and CYP7B1 (3400 SNPs), which converts cholesterol to bile may be far superior to those resulting from genotyping alone, as SNP acids. CYP5A1 is also known as thromboxane synthase (TBXAS1), a approaches may tend to overstate (or oversimplify) the biologic functionally distinct P450 that catalyzes molecular rearrangements of

TABLE 2 Summary of human P450 splice variants

Variant Disease Ratio of Viable Enzyme Family Transcript Size Exons Amino Acids Mol. Weight SNPs RNAs Links Protein

Gene no. Nucleotides kDa Total no. Total no. Total CYP1 (3) 2683–5988 3–7 512–543 58.2–60.8 49a 1052 203 0.64b (31) CYP2 (16) 1681–5024 5–9 491–596 55.7–67.6 281 11853 413 0.50 (141) CYP3 (4) 1604–2167 13–14 502–534 57.1–61.3 120 3029 93 0.47 (56) CYP4 (12) 1678–3395 5–16 505–638 59.1–71.3 170 9725 37 0.53 (90) CYP5 (1) 2093 13 594 66.9 30 5274 6 0.48 (14) CYP7 (2) 2765–2950 6 504–506 57.7–58.3 4 3688 35 0.80 (3) CYP8 (2) 3974–5632 1–10 501–527 58.1–59.5 9 1752 16 0.77 (7) CYP11 (3) 2311–3558 9 503–521 57.6–60.1 52 1807 74 0.57 (30) CYP17 (1) 2001 8 508 57.4 13 179 31 0.57 (7) CYP19 (1) 4479 10 503 57.9 47 2178 45 0.57 (27) CYP20 (1) 1811 13 470 53.4 21 1308 1 0.54 (11) CYP21 (1) 2153 10 495 56.0 30 91 35 0.52 (16) CYP24 (1) 3266 12 514 58.9 17 711 19 0.46 (8) CYP26 (3) 1569–4536 6–7 497–522 56.2–57.5 23 858 22 0.68 (16) CYP27 (3) 2579–5117 8–9 372–531 42.6–60.2 55 1582 58 0.58 (32) CYP39 (1) 2623 12 469 54.1 15 2179 2 0.50 (8) CYP46 (1) 2186 15 500 56.8 11 952 7 0.57 (6) CYP51 (1) 2817 6 860 98.0 18 388 2 0.65 (12) Total 57 1569–5632 1–16 372–860 42.6–98.0 965 48,384 1097 0.534 (515)

aTotal number of variant RNA transcripts represents the total of all nonredundant transcripts identified for human P450 forms in the AceView, Ensembl, and PubMed databases. Transcripts with the same predicted protein molecular weight (in kDa) with a fewer than four base-pair differences in nucleotide transcript length were considered the same transcript. Data for SNPs was obtained from GeneCards.org, and Disease Links were obtained from the MalaCards.org. bRatio of viable protein represents the fraction of human P450 variant transcripts predicted to code functional proteins using the highest confidence scoring metrics for the AceView (very good) and ENSEMBL (Protein coding/merged Ensembl/Havana). Total number of predicted variant proteins indicated in parenthesis. 378 Annalora et al. Downloaded from dmd.aspetjournals.org at ASPET Journals on October 3, 2021

Fig. 1. Comparative analysis of the complete human cytochrome P450 transcriptome and the total number of single-nucleotide polymorphisms. Radar plot comparison of the total number of (A) human P450 transcript variants and (B) SNPs for each of the 57 human cytochrome P450 genes are shown, on the basis of a meta-analysis of information of the NCBI’s Ensembl, PubMed, and AceView databases and the GeneCards.org website. Total transcript variants for individual P450 genes identified in this study are superimposed (in pink) over the current number listed in the AceView database alone (in purple). The expansion in variant numbers we identified highlights the challenge of predicting interindividual variability in polymorphic P450 gene splicing, predicated on existing transcript databases. Radar plot comparison of the total human SNPs associated (as reported by GeneCards.org) revealed CYP5A1 (or thromboxane synthase 1) as the most polymorphic P450 gene (5274), with CYPs 2C19 (2467), 7B1 (3400), 19A1 (2178), and 39A1 (2129) showing higher rates of polymorphism than the average P450 gene (;850 mutations per gene). (C) Bar graph representation of alternative P450 transcript-to-SNP ratios for each gene is shown at the bottom. CYP2E1, with 24 transcript variants and the lowest number of SNPs (33), displays the highest alternative transcript-to-SNP ratio (0.73) among human P450 genes. CYPs 21A2 (0.33), 27B1 (0.15), and CYP2D6 (0.11) also skew above normal for this ratio (;0.05 alternative transcripts per SNP). Improved understanding of the SNP-based mechanisms that alter P450 gene splicing will facilitate the identification of novel SNP biomarkers that are predictive of drug interactions and human disease. its substrates (Hecker and Ullrich, 1989). CYP5A1-based SNPs represent amounts of polymorphisms compared with all other human P450s, more than 10% of all P450 polymorphisms in humans and are recognized reiterating the possibility that SNP expansion among P450 genes is for altering tissue-specific splicing in blood and lung cells (Wang et al., neither random nor predictable on the basis of the tissue-specific ex- 1994), and for promoting alternative exon 12 inclusion to promote pression or -selectivity of an individual P450. What may be cerebrovascular disease (Kimouli et al., 2009). CYP2E1 displayed the more important with respect to the accumulation of SNPs is their ability lowest number of SNPs (33) and the highest alternative transcript-to-SNP to be silenced or masked via alternative or trans-splicing paradigms that ratio (0.73) among human P450 genes (see Fig. 1). CYPs 21A2 (0.33), render them invisible or inconsequential to the natural selection process. 27B1 (0.15), and CYP2D6 (0.11) also skew above normal P450 The defective nature of a P450-related SNP may depend on its ability to superfamily genes with respect to the average alternative transcript-to- manipulate the P450 transcriptome; therefore, improved understanding SNP ratio of 0.05. Improved understanding of how key SNPs interact of how key mutations alter P450 gene-splicing outcomes may be critical with the environment to alter individual P450 gene-splicing outcomes, to addressing the goal of precision medicine in the post-genomic era. therefore, could be very helpful in improving the predictive power of personalized medicine predicated on genetic screening. Therefore, some SNPs alter gene function by modulating alternative Tissue-Specific Alternative Splicing of Cytochrome P450 Genes: splicing events, but the total number in a population may be irrelevant Historical Perspective from an evolutionary perspective if they do not manifest in alternative During the late 1980s and early 1990s, several groups began to phenotypes. Furthermore, it is intriguing that several steroid hormone- appreciate the possibility that differential RNA splicing might serve as metabolizing P450s, including CYP7B1 (3400 SNPs), CYP19A1 an additional mode for regulating P450 function. In 1987, the first P450 (2178 SNPs), and CYP39A1 (2129 SNPs) display disproportionate splice variant for hepatic P450-1, later renamed CYP2C8, was reported Alternative P450 Splicing Redirects Human Drug Metabolism 379

(Okino et al., 1987). Over the next decade, several examples of alternate splicing were documented for several human P450s, including lung CYP4F2 (Nhamburo et al., 1990) and liver CYP2B6 (Miles et al., 1989; Yamano et al., 1989) and CYP2A7 (Ding et al., 1995). In the latter case, a tissue-specific, alternative splicing effect was documented, in which a 44-kDa CYP2A7-AS splice variant (compared with the 49-kDa wild- type protein) found only in 20% of liver samples was the dominant protein expressed in human skin fibroblasts. Just two years later, a similar pattern of alternative splicing behavior was documented for CYP2C18, which selectively encodes splice variants skipping different combinations of exons 4, 5, 6, and 7 in the epidermis (Zaphiropoulos, 1997). Circular RNA transcripts synthesized from the donor and acceptor sites from the skipped segments of exons 4–7 were also documented and, although the epigenetic function of this class of RNA remains unclear (Salzman et al., 2012), they were easily detected on the basis of their increased stability (Zaphiropoulos, 1997). Important studies that focused on environmentally responsive, alternative P450

splicing among xenobiotic-metabolizing P450s of the rat 2A family Downloaded from were also conducted during this era (Desrochers et al., 1996). By 1999, the CYP2C18 alternative-splicing story became even more complex when it was discovered that CYP2C18 and CYP2C19, which Fig. 2. Tissue-specific and transformation-sensitive alternative splicing of CYP1A1 cluster on 10q24 with CYP2C9, also participate in tissue- in humans. CYP1A1 is inducible in virtually every tissue of the body; however, a brain-specific CYP1A1 splice variant has been identified that preferentially skips specific, trans-splicing events, among the three genes (Zaphiropoulos, exon 6 (Kommaddi et al., 2007). This shortened form of the enzyme is spectrally

1999). Over the next decade, cell-specific splicing factors responsible for active but does not metabolize benzo(a)pyrene to toxic metabolites in brain. dmd.aspetjournals.org directing the complex, post-transcriptional gene assembly of P450s (and Structural analysis of the CYP1A1 crystal structure (PDB: 4I8V) indicates that removal of exon 6 (in yellow ribbon) would: 1) expand the opening of the pw2b other genes) were identified (Wang and Burge, 2008). However, the substrate access channel, 2) reshape the binding pocket by altering the b1-4 physiologic importance of the more unusual P450 splice variant and sheet, and 3) alter the redox partner binding surface via elimination of the K9 helix. chimeric forms remains unclear, particularly with respect to how their A similar pattern of tissue-specific, alternative exon 2 usage in CYP1A1 has been variable, tissue-specific expression alters interindividual sensitivity to reported in tumor cells (Leung et al., 2005). Ovarian cancer cells skip an 84-bp cryptic intron in exon 2 of CYP1A1 but remain in-frame to produce a catalytically hormones, pharmaceuticals, and xenobiotics. unique splice variant with diminished metabolism that localizes to the As discussed above, a single P450 gene can be translated into nucleus and mitochondria, rather than the ER. Cryptic intron removal in exon multiple, variant protein sequences, solely on the basis of tissue-specific 2 eliminates 28-amino-acid residues among helices E, F, and the E–F loop (shown in at ASPET Journals on October 3, 2021 transparent gold ribbon), which putatively expands the opening of the pw3 substrate differences in the expression of key gene-splicing machinery [e.g., small access channels located above the heme center and helix I. nuclear RNA (snRNAs)]. Several alternately spliced CYP1A1 tran- scripts identified in human brain tissues exemplify this phenomenon, as well as the concept of tissue-specific spliceosome activity (Bauer et al., structure/function and subcellular distribution observed for exon 2007). Inducible CYP1A1 is constitutively expressed in the brain, where 2-modified, CYP1A1 splice variants found both in normal and tumor it is localized to neurons in the cortex, cerebellum, and hippocampus. An tissues remains enigmatic and denotes a higher level of structural 87-bp deletion in exon 6 is observed in the brain-specific CYP1A1 form, plasticity with respect to tertiary structure than might be currently but not in liver (Chinta et al., 2005). Human brain tissues expressing the appreciated or expected from classic structural studies of wild-type Dexon6-CYP1A1 variant do not express wild-type CYP1A1 and they P450 forms (Johnson and Stout, 2013). metabolize known substrates [e.g., benzoxy-resorufin and benzo(a)pyrene Perhaps the most dramatic, and mutually exclusive, example of tissue- (BP)] at different rates than wild-type enzyme (Kommaddi et al., 2007). specific, alternate-exon usage in P450s was documented for CYP4F3, Furthermore, cells expressing wild-type CYP1A1 generate DNA adducts which can be expressed as two distinct splice variant forms (CYP4F3A via the formation of reactive 3-OH-BP products, whereas cells expressing and CYP4F3B). These two CYP4F3 isoforms differ only by their the Dexon6-CYP1A1 variant in brain do not (Kommaddi et al., 2007). variable usage of exons 3 and 4, which encode distinct portions of the active On the basis of the structural analysis of CYP1A1 in Fig. 2 (derived site and substrate access channel, allowing them to fine-tune substrate from PDB: 4I8V; Walsh et al., 2013), tissue-specific, alternative specificity (Christmas et al., 1999, 2001). CYP4F3A is expressed in blood splicing of exon 6 may reshape the size and polarity of the CYP1A1 and bone marrow myeloid cells, selectively expresses cassette exon 4 (and substrate access channel to alter substrate recruitment and recognition not 3), and has low affinity for B(4) (LTB4). In contrast, processes and redox-partner binding interactions. Exon 6 of CYP1A1 CYP4F3B, expressed exclusively in the liver and kidney, encodes exon appears to be organized for “plug and play” utilization in the brain, 3 (and not 4) and has high affinity for LTB4, in addition to other where targeted skipping does not compromise the global P450 fold but functional differences. The highly concerted, tissue-specific splicing alters substrate specificity for a spectrum of endogenous substrates, of CYP4F3 exemplifies the functional utility of the modular P450 including . This tissue-specific exon 6 usage of CYP1A1 gene platform, one that allows cells to sample a continuum of gene is complemented by a similar pattern of alternative exon 2 usage found products as dictated by tissue-specific physiologic stress conditions. in human endothelial cells, leukocytes, and tumor cells (Leung et al., However, it should also be clearly noted that not all P450 genes are 2005, Bauer et al., 2007). As shown in Fig. 2, targeted skipping of an divided into an equal number of coding and noncoding regions, which 84-bp cryptic intron in exon 2 of CYP1A1 produces a catalytically suggests that the accumulation of introns may be an adaptive and ongoing unique splice variant with diminished estradiol metabolism that process. For example, the CYP1B1 gene, which encodes a P450 enzyme localizes to the nucleus and mitochondria rather than the endoplasmic that metabolizes both xenobiotics and endogenous substrates, con- reticulum (ER) (Leung et al., 2005). The added diversity in both tains only two coding exons (Supplemental Fig. 2) and, therefore, is 380 Annalora et al. not subject to the same patterns of alternative splicing as other CYP1 family members 1A1 and 1A2, which have six coding exons (Stoilov et al., 1998). In this regard, the most well studied splice variants of CYP1B1 all represent severe truncations or deletion mutants, not exon skipping (Tanwar et al., 2009), and CYP1B1 retains an unusual, elongated C-terminus, further distinguishing it from other human P450 forms. The CYP1B1 gene is also uniquely located on at locus 2p21– 22, which in humans is derived from an ancient fusion of chimpanzee 2a and 2b (Faiq et al., 2014). It is difficult to speculate why the CYP1B1 gene is organized so differently from closely related CYP1A genes located on chromosome 15. However, observations linking a common set of nuclear proteins to both the alternative splicing process and NMD seem to imply that expansion of gene complexity may be related more to the rate at which a given mRNA transcript is processed by the spliceosome and ribosome, rather than other evolutionary factors (Lejeune and Maquat, 2005; McGlincey and Smith, 2009). Ultimately, the adaptive forces driving the modular organization of

genes into variable numbers of introns and exons remain a point of Downloaded from speculation. However, a gene’s intronic complexity clearly influences Fig. 3. Alternative-splicing of the human CYP24A1 gene: tissue- and tumor- specific exons 1, 2, and 10 inclusion. CYP24A1 is a mitochondrial P450 composed its sensitivity to alternate-splicing events and the domain swapping of 11 coding exons that metabolizes the vitamin D hormone to regulate its role in paradigm. The tissue-specific use of alternative promoters and non- calcium homeostasis, cell growth, and immunomodulation. Alternatively spliced AUG translation start-sites also is facilitated by this type of hidden transcript variants have been identified for this gene, including a CYP24A1 splice variant (CYP24sv) specifically expressed in macrophage (Ren et al., 2005), which gene complexity, which allows discrimination among a set of well skips exons 1 and 2. The 372-amino-acid variant protein (;43 kDa) lacks the 9 9 defined exons and poorly defined pseudoexons. N-terminus, helices A , A, B, and B and portions of the b1 sheet but probably dmd.aspetjournals.org In 2005, a truncated, CYP24A1 splice variant incapable of metabo- retains the ability to bind heme and substrate in some capacity. Ren and coauthors lizing hormonal forms of vitamin D was identified in the human concluded that CYP24sv represents a cytosolic variant with dominant-negative function that quarantines 25-hydroxyvitamin D3 and other hormonal forms of macrophage (Ren et al., 2005). CYP24A1 is the prototypical mito- vitamin D, slowing the rate of their metabolism in the mitochondria or endoplasmic chondrial P450 responsible for the side-chain cleavage of the vitamin reticulum. Structural analysis of CYP24A1 (PDB: 3K9V) suggests that CYP24sv’s D hormone and it possesses a mitochondrial localization sequence in unique N-terminus, derived from intron 2 (shown in green), reshapes the pw2a substrate access channel and provides contacts for sealing the ligand binding pocket the first 30 residues of its N-terminus (Annalora et al., 2004). The via interactions with helices F–G and the b1 and b4 sheets systems. It is notable that CYP24A1 splice variant found in activated macrophage (CYP24A1- exons 1 and 2 exclusion in CYP24A1 is exacerbated in human tumors of the breast SV1) lacks 153 N-terminal residues encoded by exons 1 and 2, and and colon, where N-terminally truncated splice variants of approximately 40, 42, and at ASPET Journals on October 3, 2021 gains eight residues preceding the content from exon 3, via a partial 44 kDa have been identified (Fischer et al., 2009a; Horváth et al., 2010; Scheible et al., 2014). An additional, prostate cancer-related, splice variant of CYP24A1 that insertion of intron 2. CYP24A1-SV1 is thought to function as a soluble cleanly skips exon 10 has also been described (Horvath et al., 2010; Muindi et al., variant that disrupts normal vitamin D hormone metabolism via the 2007). Exon 10 encodes much of the protein’s proximal surface, including the b1–3 sequestration of key intermediates from both wild-type CYP27B1 and sheet, meander region, CYS loop, and portions of the L-helix involved in heme- thiolate bond formation. Variants lacking exon 10 may express defects in heme and CYP24A1 (Ren et al., 2005). In this regard, the CYP24A1-SV1 variant redox partner binding, giving rise to an alternative dominant negative form that may may promote a discrete pattern of vitamin D hormone accumulation in retain membrane-binding features; loss of exon 10 could also refine the alternative the macrophage, and potentially other tissues such as the intestine, where functions of cytosolic splice variants lacking exons 1 and 2. vitamin D hormonal leakage from irritated cells is thought to contribute to the burst of extracellular hormone observed in inflammatory disorders like Crohn’s disease (Abreu et al., 2004; Mangin et al., 2014). A regulation, particularly for variants subject to atypical subcellular structural analysis of the CYP24A1-SV1 variant is shown in Fig. 3 (on trafficking events. the basis of PDB: 3K9V; Annalora et al., 2010), and it highlights how Therefore, although increasingly common in the literature, the the targeted loss of N-terminal residues from exons 1 and 2 could re- functional significance of many tissue-specific P450 variants remains configure the membrane-binding surface and substrate access channel to elusive. CYP2C8, for example, is constitutively expressed in the liver alter the catalytic properties of wild-type CYP24A1. at 5–7% of the total tissue P450, has more than 20 known alternately Interestingly, the orphan P450 CYP27C1, a close relative of spliced variants, and is a breast cancer biomarker owing to its high CYP24A1, encodes a 372-residue wild-type protein that resembles the expression in mammary tumors (Knüpfer et al., 2004). Bimodal CYP24A1-SV1. CYP27C1 lacks a prototypical N-terminus and initiates targeting to both mitochondria and ER have been reported for coding in the middle of canonical helix C, at a position analogous to CYP2C8 and several other P450s, including CYP1A1, 1B1, 2B1, the alternative start site used by CYP24A1-SV1 (Ren et al., 2005). 2E1, and 2D6. Although it is clear that alternative splicing directs this CYP27C1 is expressed in liver, kidney, pancreas, and several other process in some tissues, proteolytic cleavage of N-terminal targeting tissues, and remained an orphan for over a decade (Wu et al., 2006), until sequences can also produce this effect (Bajpai et al., 2014). One 2C8 recently, when it was shown to mediate the conversion of vitamin A1 variant (variant 3; ;44 kDa) with an alternative N-terminus is (all-trans retinol) into vitamin A2 (all-trans 3,4-dehydroretinal) in cell targeted to the mitochondria, where it may contribute to oxidative culture (Kramlinger et al., 2016). It is intriguing that CYP27C1 may stress (Bajpai et al., 2014). The catalytic competency of these represent a codified version of the more soluble P450 isotype re- untethered variants remains a topic of intense debate, as it is often capitulated by CYP24-SV1, and that this transition may mediate a difficult to assign an orphan function to a variant protein that has lost general switch between vitamin A and vitamin D substrate specificity. selectivity to the gene’s prototypical substrates. A review of P450 Improved knowledge of CYP27C1 structure/function could help to splice variants subject to alternate subcellular trafficking is shown in unravel some of the complexity associated with the variant P450 Supplemental Table 1. Computational analysis from the AceView Alternative P450 Splicing Redirects Human Drug Metabolism 381 database (Thierry-Mieg and Thierry-Mieg, 2006) indicate that some intronic SNPs may facilitate the alternate splicing pattern seen for P450 variants may be targeted to the nucleus, peroxisome, plasma CYP24A1 in prostate cancer cells, vitamin D hormone exposure also membrane, or vacuole compartments, in addition to classic targets in putatively alters splicing via discrete interactions among the vitamin D the ER, mitochondria, or cytoplasm. This analysis hints that P450 receptor (VDR) and nuclear-splicing factors like the heterogeneous splice variants, including tumor-specific forms, may be performing nuclear ribonucleoprotein C (hnRNPC; Zhou et al; 2015). Tumor cells an array of unrecognized functions, including diverse roles at the cell overexpressing CYP24A1, therefore, may not retain sufficient hormone surface and beyond, which are rarely considered (Eliasson and to activate the VDR properly, disrupting normal gene-splicing events Kenna, 1996; Stenstedt et al., 2012). needed to properly transduce the hormone’s immunogenic, prodiffer- entiation properties. CYP24A1 splice variants have now been docu- mented among multiple human breast tumor cell lines (e.g., MCF-7 and Transformation-Sensitive Alternative Splicing in the Cytochrome MCF-10) and in both healthy and malignant breast tumor tissues. In P450 Superfamily benign tissue, wild-type CYP24A1 (56 kDa) was the only isoform This transformation-specific, P450 splicing phenomenon was first present. In malignant tissues, three splice variants (40, 42, and 44 kDa) documented in 1996 for the prototype P450, CYP19A1 (or aromatase), were expressed, similar to cultured MCF-7 breast tumor cells that which facilitates estrogen biosynthesis via three successive hydroxyl- express the 42- and 44-kDa variants (Fischer et al., 2009a; Scheible ations of the androgen A ring and displays a unique pattern of exon et al., 2014). A similar pattern of transformation-sensitive splicing is 1 (and cryptic intron) usage in human breast tumor cells and tissues seen in human colon cancer tissues, where the histological grade and the

(Zhou et al., 1996). A similar pattern of CYP19A1 exon 1 variant ex- gender of the patient modulates the formation of the three detectable Downloaded from pression also has been documented in sheep, cattle, and rabbit, suggesting CYP24A1 splice variants (Horváth et al., 2010). Peng and coworkers this paradigm is not exclusive to humans or transformed-tissue types (2012) further refined our understanding of this process in the colon by (Vanselow et al., 1999; Bouraïma et al., 2001). More recently, it was demonstrating that cell- or tissue-specific splicing patterns were sup- determined that CYP19A1’s exon 5 also is defined poorly and subject to pressed by parathyroid hormone signaling, but only in the absence of the both splicing mutations and physiologic alternative splicing events (Pepe vitamin D hormone, which may be the master regulator of both a et al., 2007). Skipping of exon 5, which encodes complete -helices D and CYP24A1 gene expression and splicing. dmd.aspetjournals.org E in CYP19A1, eliminates prototypical P450 aromatase activity in both The phenotypic instability exemplified by the CYP24A1 gene in steroidogenic and nonsteroidogenic tissues (Lin et al., 2007; Pepe et al., human tumors complicates the therapeutic landscape from which 2007). These observations suggest a tissue-specific, regulatory mechanism rational drugs for cancer can be conceived (Trump et al., 2006). for aromatization on the basis of alternative exon 5 inclusion in humans. Epigenetic modification of the CYP24A1 gene and its promoter also A structural analysis of this CYP19A1 splice variant is depicted in may contribute to tissue-specific differences in P450 expression Supplemental Fig. 3 (on the basis of PDB: 3S79; Ghosh et al., 2012). (Johnson et al., 2010). Frontiers in this area of research will likely In aromatase, exon 5 skipping naturally suppresses androgen metab- address the coordinated role that nuclear hormone receptors and olism by redefining the determinants of substrate recognition. While epigenetic splicing factors (including noncoding forms of RNA) play at ASPET Journals on October 3, 2021 transfection studies suggest CYP19A1 variant proteins are encoded in managing both the transcriptome and the epigenome. and not subject to NMD, their ability to alter the safety and efficacy of It should be noted that the CYP27B1 gene, which is highly related to several CYP19A1 (aromatase) inhibitor drugs [e.g., Arimidex (anastro- CYP24A1, was also recognized early as being sensitive to transformation- zole), Aromasin (exemestane), Femara (), and Teslac (testolac- specific alternative splicing in malignant human glioma cells (Maas et al., tone)] remains unclear (Hadfield and Newman, 2012) and an area of 2001). CYP27B1 variant expression has now been documented in tumors active investigation (Liu et al., 2013a; Liu et al., 2016). of the breast, skin, cervix, kidney, and ovaries (Diesel et al., 2004; Fischer The tissue-specific, alternative start-site usage of CYP24A1, dis- et al., 2007, 2009b; Wu et al., 2007). Tissue-specific CYP27B1 splicing cussed above, appears to be exacerbated in several human transcript also is common in healthy skin, where a 59-kDa variant with a 3-kDa variants in which an array of truncated splice variants of approximately insertion between exons 2 and 3 and exclusive to skin was observed 40, 42, and 44 kDa have been identified with similar N-terminal and linked to the activity of cAMP response element–binding elements modifications (Fischer et al., 2009a; Horváth et al., 2010; Scheible et al., (Flanagan et al., 2003). Fluctuations in skin cell density, calcium con- 2014). An additional, cancer-related, splice variant of CYP24A1 that centrations, and UV-B light exposure were also shown to affect the cleanly skips exon 10 has also been documented (Muindi et al., 2007; splicing process (Seifert et al., 2009). Collectively, these findings help to Horvath et al., 2010). In CYP24A1, exon 10 encodes much of the validate our core hypothesis that alternative gene splicing, dictated by protein’s proximal surface, including the CYS loop, and a portion of both cellular and environmental factors such as UV light exposure and helix L involved in heme-thiolate bond formation and adrenodoxin nutritional status, can alter a patient’s metabolic profile in tissue-, age-, recognition (shown in Fig. 3). Although the function of this cancer- sex-, exposure-, and disease-specific ways that are difficult to predict. specific variant remains unknown, it may have reduced ability to bind Research focused on CYPs 19A1, 24A1, and 27B1 is among the most heme and redox partners compared with truncated variants (skipping comprehensive on transformation-sensitive alternative splicing in human exons 1 and 2), and thus may represent an alternative, dominant-negative P450s, and the literature is replete with anecdotal reports of alternative variant also capable of modulating vitamin D hormone metabolism and P450 splicing among various transformed cell types and tissues. For metabolite trafficking at the plasma membrane level. As discussed example, CYP2E1 transcripts that selectively skip exons (2, 2–3, and previously, the cellular mechanisms regulating CYP24A1 alternative 2,4,5–59,6) were identified in lung carcinoma cell lines but not in spicing in the macrophage remains unclear, although cell-specific corresponding lung tumor tissue or liver extracts, where splice variants variations in splicing factors (e.g., heterogeneous ribonuclear protein were found to be noninducible by ethanol exposure (Bauer et al., 2005). A1) are thought to be responsible (Ren et al., 2005). A more complex The authors noted at the time that none of these splice variants were pattern of “transformation-sensitive” alternative CYP24A1 gene splic- accounted for in the National Institutes of Health (NIH) AceView ing also has been documented in human tumors of the prostate (Muindi database, highlighting the longstanding challenge associated with et al., 2007), breast (Fischer et al., 2009a; Scheible et al., 2014), and cataloging splice variant forms derived from cell-specific abnormalities colon (Horváth et al., 2010; Peng et al., 2012). It is thought that whereas versus those generated from normal, tissue-specific splicing paradigms. 382 Annalora et al.

Furthermore, several orphan P450s, including CYP1B1, CYP2S1, and The CYP2D gene family also is complex, clustered, and subject to CYP2W1 are highly associated with specific human tumor types, making trans-splicing events; it is composed of CYP2D6 and 4 pseudogenes them important prodrug targets for chemoprevention strategies (Stenstedt (CYP2D7P1 and 2 and CYP2D8P1 and 2). Alternative splicing of the et al., 2012). Unfortunately, as discussed above, the alternative splicing CYP2D family pre-mRNAs has been detected in human liver, breast, behavior of orphan P450s is often as poorly understood as are their and lung tissue (Huang et al., 1996, 1997), including a highly expressed cryptic functions. splice variant that completely skips exon 6. As shown in Supplemental Interestingly, orphan CYP2W1, which is expressed in human tumor Fig. 4B, analysis of the CYP2D6 crystal structure (PDB: 4WNU: Wang types of the colon, is associated almost exclusively with aberrant P450 et al., 2015) reveals that exon 6 encodes the complete coding sequence behavior, including an inverted orientation in the ER membrane, and for helix I, and that skipping this important region would dramatically glycosylation-sensitive trafficking to the plasma membrane (Gomez alter protein structure/function by removing key catalytic residues (e.g., et al., 2010; Stenstedt et al., 2012). The increased expression of Thr-309) and remodeling the distal pocket of the . Alternate CYP2W1 in tumors is associated with demethylation of a CpG island CYP2D6 exon usage is tissue-selective, and variant forms skipping exon in the exon 1-intron 1 junction of the gene. How this mechanism relates 3 and portions of exon 4 have been documented that alter enzyme to the paucity of normal CYP2W1 expression in healthy tissues remains function and subcellular localization (Sangar et al., 2010). CYP2D6 unclear, but it implies that the expression of some specialized P450s may polymorphisms are linked to modified enzyme activity and may only be invoked under extreme cellular stress, when an alternative P450 serve as biomarkers for poorly metabolizing phenotypes sensitive to activity may be critical for balancing cellular homeostasis. The abnormal dose-dependent drug toxicity. Recently it was reported that intronic

expression and trafficking of CYP2W1 to the plasma membrane could polymorphisms in CYP2D6*41 individuals (considered intermediate Downloaded from promote an autoimmune response as part of its mechanism. A similar metabolizers) could dramatically increase the expression levels of autoimmune reaction is associated with aberrant CYP2E1 trafficking in CYP2D6 variants lacking exon 6 (Toscano et al., 2006). These animal tissues treated with halothane (Eliasson and Kenna, 1996). The findings, coupled with the complex organization of the CYP2D gene NIH’s AceView and Ensembl (Cunningham et al., 2015) databases cluster, indicate that both alternative and trans-splicing mechanisms indicate that CYP2W1 encodes at least 11 transcript variants, three of may underlie the complex genotype-phenotype relationships asso-

which lack one or more exons and are associated with hepatocellular ciated with variable CYP2D6 metabolism in humans, and that dmd.aspetjournals.org carcinomas, adenocarcinomas, and neuroblastomas. Nearly 200 of the CYP2D6 is well organized structurally to exploit both tissue- and 965 alternatively spliced P450 transcripts we identified in public database transformation-selective splicing mechanisms targeting helices C searches were associated with a well-characterized human tumor type. and D (exon 3) and helix I (exon 6). Improved understanding of their tumor-specific cellular roles and the The CYP3A gene family (comprising CYPs 3A4, 3A5, 3A7, and mechanisms regulating their induction should aid in the identification of 3A43) is also highly complex in humans, with each gene consisting of improved disease biomarkers and P450 variant-specific therapeutic agents. 13 exons (with ;71–88% amino acid identity) that cluster on chromo- some 7q21–22.1, where the CYP3A43 gene is in a head-to-head orientation with the other three genes (Finta and Zaphiropoulos, 2000). at ASPET Journals on October 3, 2021 Trans -Splicing of Cytochrome P450 Gene Chimeras Several chimeric forms of CYP3A mRNAs have been described, in It is well understood that both transformation-sensitive cellular changes which CYP3A43 exon 1 is joined at CYP3A4 and CYP3A5 canonical and P450 polymorphisms can alter protein expression in multiple ways splice sites, implying a trans-splicing mechanism that bypasses classic that are often difficult to predict from primary sequence analysis alone. transcriptional control paradigms. In Supplemental Fig. 5A, a structural This challenge is exacerbated in situations where chimeric P450 genes analysis of CYP3A4 (on the basis of PDB: 4K9T; Sevrioukova and are created via trans-splicing events among extended (or multiple) Poulos, 2013) reveals the highly complex segmentation of the CYP3A4 pre-mRNA transcripts. Therefore, although mutations in a gene like gene into 13 exonic domains, which, as described above, may potentiate CYP2C9 are linked to poor metabolism of several drugs, including rapid translation upon induction (Lejeune and Maquat, 2005; McGlincy and , the role that alternative or trans-gene and Smith, 2008). Primary amino acid sequence alignments of key splicing plays in manifesting a deleterious CYP2C9-related metabolic CYP3A genes suggest exon 1 may encode distinct transmembrane anchor phenotype are rarely considered. For example, several CYP2C9 splice helices and membrane-binding architecture (Supplemental Fig. 5B), variants (CYP2C9sv) have been identified (Ohgiya et al., 1992), hinting that CYP3A chimeras may encode a spectrum of phenotypes with including a liver-specific form that skips exon 2 (Ariyoshi et al., unique tissue-specific distribution and functionalities. A CYP3A4 trans- 2007). The internally truncated CYP2C9 variant is spectrally active splicing variant containing CYP3A43 exon 1, followed by CYP3A4 but does not metabolize prototype CYP2C9 substrates. A structural exons 4–13, has been described; it retains detectable testosterone 6-b- analysis of the CYP2C9 crystal structure (PDB: 4NZ2; Brändén et al., hydroxylase activity despite losing membrane-binding features encoded 2014; shown in Supplemental Fig. 4A) hint that removal of exon 2 by exons 2 and 3, comprising helices A9 and A, and portions of the b1-1 would reshape portions of the reference protein relative to its membrane- sheet (Supplemental Fig. 5C). A CYP3A4 trans-splicing variant binding surface, substrate access channel, active site, and redox partner containing CYP3A43 exon 1, followed by CYP3A4 exons 7–13, has binding surface. CYP2C9 is located within a cluster of P450 genes on been studied also, and it displays minimal 6-b-hydroxylase activity chromosome 10q24 and is subject to nonrandom, trans-splicing events (Supplemental Fig. 5D). This story is further complicated by the presence with neighboring CYPs 2C8, 2C18, and 2C19 in liver and skin, where of additional CYP3A pseudogenes (CYP3AP1 and CYP3AP2) in the CYP2C18exon1–like sequences were found spliced into different intergenic regions between CYP3A4, 3A7, and 3A5, where discrete combinations of introns and exons from the CYP2C9 genes (Warner exons from the pseudogenes also have been found trans-spliced into et al., 2001). Common 2C9-related SNPs linked to various metabolic CYP3A7 transcripts (Finta and Zaphiropoulos, 2002). Ultimately, disorders, therefore, may only function to modulate the innate splicing CYP3A4 and CYP3A5 splicing complexity may help to explain how process indirectly. This is particularly true if their coding regions are intronic SNPs in CYP3A4*22 patients alter tacrolimus metabolism, spliced out of chimeric 2C forms being expressed on an interindivid- without introducing a nonsynonymous mutation to an exon (Elens ual basis, highlighting another major flaw in GWAS targeting of the et al., 2011). The predictive power of GWAS may therefore need to be components of gene clusters. reconsidered, particularly with respect to the most complex P450 gene Alternative P450 Splicing Redirects Human Drug Metabolism 383 families defined by the presence of pseudogenes (e.g., CYP 2C, 2D, proteins) may account for interindividual variability in recognizing 3A, 21A, and 51A families) and trans-splicing mechanisms. the CYP3A5*3 SNP, there are currently no models to explain how this complex, salt-sensitive splicing phenomenon is being regulated in the kidney. The alternative splicing of CYP3A5 variant exem- SNP-Sensitive Alternative Splicing in the Cytochrome P450 plifies why SNP-based approaches to personalized medicine are so Superfamily challenging to implement, when the physiologic impact of a given As discussed above, tissue-specific, alternative and trans-splicing SNP may be the opposite of what is expected. Computational approaches behaviors have now been documented for several important P450 gene that consider a patient’s age, genotype, nutrition, and disease status may families; however, SNPs can destabilize this process in complex ways ultimately be needed to make good predictions concerning the impact of that are often difficult to predict. SNPs that target cryptic intronic a given SNP on an individual’s transcriptome. recognition elements or discrete intron/exon splice junction boundaries The challenge of addressing SNPs for personalized medicine is further can alter translation start-and-stop site usage, and the expression ratios complicated by observations that some xenobiotics, including amino- among wild-type and variant P450 transcripts. Some P450 SNPs are glycosides and cyclohexamides (Busi and Cresteil, 2005), can alter the exceedingly rare, occurring in less than 1% of the population. These rare proofreading capabilities of the ribosome, allowing inhibition of pre- mutations are linked to congenital defects, including glaucoma (CYP1B1; mature stop codons and translational read-thru of alternate transcripts that Stoilov et al., 1998; Tanwar et al., 2009; Sheikh et al., 2014), 17-a may or may not remain in-frame with respect to the reference transcript. hydroxylase deficiency (CYP17A1; Yamaguchi et al., 1998; Costa- Improved knowledge of a patient’s chemical exposure history and

Santos et al., 2004; Hwang et al., 2011; Qiao et al., 2011), congenital epigenetic status, therefore, may become increasingly important when Downloaded from adrenal hyperplasia (CYP21A2; Robins et al., 2006; Lee, 2013; Szabó making predictions about food and drug safety. Furthermore, personal- et al., 2013; Sharaf et al., 2015), spina bifida (CYP26A1; Rat et al., ized approaches to medicine solely on the basis of SNPs identified via 2006), focal facial dermal dysplasia (CYP26C1; Slavotinek et al., 2013), cohort studies or GWAS may be fundamentally flawed and potentially and cerebrotendinous xanthomatosis (CYP27A1; Garuti et al., 1996; misleading, in that they fail to account for the milieu of compensatory Verrips et al., 1997; Chen et al., 1998; Tian and Zhang, 2011). Other cellular mechanisms that are adept at masking the most deleterious effects

P450 polymorphisms that alter splicing are associated with neurologic of even the most highly penetrant mutations. dmd.aspetjournals.org and metabolic diseases, including Parkinson’s disease (CYP2D6, Denson et al., 2005; CYP2J2, Searles Nielsen et al., 2013), hypertension (CYP4A11, Zhang et al., 2013; CYP17A1, Wang et al., 2011), breast Addressing Nontraditional P450 Function and Trafficking Events cancer (CYP2D6, Huang et al., 1996; CYP19A1, Kristensen et al., 2000;) Our meta-analysis and review of splice variability within the colon cancer (CYP2W1,Stenstedt et al., 2012), and lung cancer cytochrome P450 superfamily has revealed an under-appreciated (CYP2D6, Huang et al., 1997; CYP2F1, Tournel et al., 2007). However, level of alternative transcript expansion and variant protein structural the complex etiology of these disorders can make it difficult to interpret complexity that is poorly addressed by conventional P450 structure/ the exact role that a P450 SNP might play in the actual onset or function paradigms. Well studied splice variants from the CYP1A at ASPET Journals on October 3, 2021 progression of a disease. Multiple studies of various intergenic, intronic, family reveal a sophisticated domain-swapping mechanism that and exonic polymorphisms that alter human drug and xenobiotic allows for tissue-specific sampling of alternative protein conforma- metabolism have also been reported (CYP1A1, Allorge et al., 2003; tional space. Discrete arrays of tissue-specific snRNAs (U1–U9) and CYP2C19, de Morais et al., 1994; Ibeanu et al., 1999; Satyanarayana ancillary splicing factors can alter the biologic syntax of a gene, thus et al., 2009; Sun et al., 2015; CYP2D6, Toscano et al., 2006; Lu et al., providing a granular mechanism for this natural phenomenon that is 2013; Wang et al., 2014; CYP3A4, Elens et al., 2011; and CYP3A5, highly responsive to environmental factors, including diet and chemical Kuehl et al., 2001; Busi and Cresteil, 2005; Lee et al., 2007; García- exposure. In human brain, the alternative splicing of CYP1A1 appears Roca et al., 2012). In many cases, the most pharmacologically relevant linked to a tissue-specific sensitivity to a reactive P450-product (e.g., SNPs are those affecting the innate splicing behavior of a given gene. 3-hydroxybenzo[a]pyrene; Kommaddi et al., 2007). How this gene- This observation highlights why knowledge of a patient’s discrete specific sensitivity is transduced and imprinted at the genetic level P450 transcriptome is so important when selecting a personalized remains enigmatic, but the cellular microenvironment clearly dictates medicine regimen. what hidden structural complexity of a P450 gene may be exploited. It is now clear that numerous P450-related SNPs are found between This observation helps explain why some P450 variants, like CYP24A1- genes and exons, or in the 59 or 39 untranslated regions of RNA SV1, which are normally expressed in the immune system, can become transcripts. Polymorphisms in this class have now been identified for major players in tumor cells, where they may elaborate a common virtually every human P450 isoform, with each having the potential emergency function, such as the suppression of vitamin D hormone to alter splicing in unique ways in different individuals. For example, metabolism. This cellular phenomenon corresponds well with related the CYP3A5*3 SNP (rs776746, also known as 6986A.G) intro- vitamin D hormone–scavenging strategies, such as the 3-epimerization duces a mutation in intron 3 that alters normal splicing of exon 4, pathway, which can be induced to modify the vitamin D hormone’s producing a nonfunctional, truncated CYP3A5 protein that predom- chemical ring structure to limit its efficient catabolism via CYP24A1 inates in many Caucasians (Kuehl et al., 2001). Expression of the (Rhieu et al., 2013). To date the enzyme responsible for the 3-epimerization wild-type CYP3A5*1 protein is highly variable among populations of the vitamin D hormone remains unknown (Bailey et al., 2013). andislinkedtoanincreasedsensitivitytosalt-inducedhypertension. New appreciation for the increasingly diverse and complex roles that Interestingly, some individuals who are homozygous CYP3A5*3/*3 P450 splice variants play in normal biology should help reinvigorate can express both the truncated variant and properly spliced, wild-type discussions of P450 gene structure and evolution, particularly with CYP3A5 (Lin et al., 2002). Because the CYP3A5*3 mutation occurs respect to the selective forces that regulate P450 gene duplication and in intron 3, over 100 base pairs upstream from the splice donor site of mutation events. Although the origin and expansion of spliceosomal exon 4, it is unclear how this SNP alters normal recognition of the introns remains highly debated (Rogozin et al., 2012; Qu et al., 2014), intron 3-exon 4 junction. Although variations in the expression of the intron-exon boundaries of 17 of 18 mammalian P450 families appear ubiquitous splicing factors (e.g., U1 small nucleolar ribonuclear to be well conserved across almost 420 million years of evolution, as 384 Annalora et al. evidenced by the fugu (pufferfish) genome (Nelson, 2003); only the splicing regulatory-factor proteins) and alter RNA polymerase (Pol) II CYP39 gene is missing from fugu and all known fish. The bulk of P450 processivity. Furthermore, because some introns are defined by AU-AC structural diversity encoded by the human genome, therefore, likely junctions rather than GU-AG junctions, the splicing of these introns may predates the divergence of tetrapods from ray-finned fish over 420 million be less sensitive to ROS-mediated events and, therefore, may be subject years ago. In this regard, brain-selective expression of CYP1A1 variants to a different suite of regulatory-splicing mechanisms. For example, the lacking exon 6 in humans may be no more evolved than tissue-specific induction of environmentally responsive genes like adenosine-deaminase expression of CYP19A1 splice variants found in fugu. In each case, the could alter the recognition of a prototypical AU-AC junction in a induction of P450 splice variants appears to provide a novel mechanism similar way, via adenosine-to-inosine (A-to-I) RNA editing (Solomon for modulating steroid hormone pleiotropism among a diverse set of et al., 2013). Even mitochondrial genes lacking spliceosomal introns target tissues. could be affected by this phenomenon, as the discrete dinucleotide The selective pressures that created the modular framework of the sequences that coordinate their autocatalytic, self-splicing events P450 gene, therefore, likely predate the Cambrian explosion 540 million could also be potentially altered by P450-mediated ROS modulation years ago, and lurk somewhere in the history of deuterostome evolution (Zimmerly and Semper, 2015). (Nelson, 2003). However, contemporary analyses of P450 splicing mechanisms are needed to help elaborate the relationship between alternative subcellular trafficking and alternative P450 variant function Shaping an Improved Roadmap toward Precision Medicine in the mitochondria, nucleus, peroxisome, cytoplasm, or plasma Because changes in mitochondrial function can alter global alternative

membrane (Supplemental Table 2). Because alternative splicing and splicing events, it is not surprising that both phenomenon are linked to Downloaded from alternative start-site usage can generate even greater P450 structural the induction of cellular heterogeneity and human disease pathology diversity than those predicted to arise from post-translational modi- progression (Raj and van Oudenaarden, 2008; Hanahan and Weinberg, fications of intracellular targeting motifs, the mechanisms underlying 2011; Pagliarini et al., 2015). Despite a growing appreciation for the role their emergence and regulation deserve greater attention. For example, that alternative splicing plays in promoting phenotypic variability, the if subtle differences in the membrane-binding features of CYP1A1 and identification of genetic polymorphisms linked to disease, which may or

CYP1A2 can modulate redox partner recruitment and protein interactions may not alter gene-splicing events, remains a core focus of pharmaco- dmd.aspetjournals.org within ordered and disordered regions of the lipid bilayer (Park et al.; genomics. Mutations in single genes have now been identified for over 2015), metamorphic structural changes associated with some P450 4000 human diseases, of which 5–15% are the result of SNPs resulting in variants may underlie an even greater array of uncharacterized (and nonsense mutations (Mort et al., 2008). However, SNPs occur in coding potentially moonlighting) P450 functions (Zhao and Waterman 2011; regions of genes with a frequency of approximately 1.5%, and of those, Lamb and Waterman, 2013). only one-third are expected to result in nonsynonymous mutations and a As the landscape of alternate P450 gene functions continues to much smaller number alter pre-mRNA processing, leading to a frequency expand, the cellular basis for alternatively targeting microsomal in change of phenotype of only ;0.5%. When one considers that humans P450 forms to the mitochondria (e.g., CYP1A1, CYP1A2, CYP1B1 express almost 50,000 SNPs across the 57 human P450 genes, fewer than at ASPET Journals on October 3, 2021 CYP2B1, CYP2B2, CYP2E1, CYP3A1, and CYP3A2) is still being 250 may be capable of significantly altering a patient’s metabolic investigated (Anandatheerthavarada et al., 1997). However, a putative phenotype, an insight we contend may help streamline PGx approaches role in modulating destructive, subcellular reactive oxygen species to personalized medicine (Zhou et al., 2009; Zanger and Schwab, 2013). (ROS) production has recently been proposed (Dong et al., 2013). For example, although CYP2D6 genotyping is no longer recommended This theory emerges from evidence that the alternative trafficking of in the clinical setting for treatments (Abraham et al., 2010; the related NADPH:quinone oxidase-1 (NQO1) gene, which limits Wu, 2011; Lum et al., 2013), the FDA still considers CYP2D6 clinically ROS by preventing semiquinone redox cycling, can protect the actionable from a PGx perspective for codeine and other drugs (Crews mitochondria from stress-induced oxidative damage. P450 variants et al., 2014). Unfortunately, over 74 allelic variants of CYP2D6 have may potentially play a similar role by redirecting the metabolism of already been described, which greatly complicates genetic testing and the reactive subcellular compounds. However, because P450s can also clinician’s ability to select the appropriate pharmacotherapy and dose become uncoupled and directly produce ROS via the release of (Zhou, 2009). Fortunately, organizations such as the Clinical Pharma- reactive products (i.e., via uncoupled Fenton-type reactions), further cogenetics Implementation Consortium (CPIC) and the Pharmacoge- attention to the role of alternative P450 splicing in the modulation of nomics Knowledgebase (PharmGKB) are working to standardize mitochondrial stress and dysfunction seem warranted. In this regard, it methodologies for PGx data analysis and clinical guidance for specific is intriguing that many P450 splice variants that undergo alternative gene-drug pairings (Caudle et al., 2016). However, CPIC concedes trafficking often lack a significant portion of the membrane binding that some rare P450 variants may not be included in genetic tests, and domain and/or substrate-access architecture (typically encoded by that patients may be assigned “wild-type” genetics by default (Crews exons 1–3), which may expand heme accessibility or the rate of et al., 2014). When paired with additional uncertainties regarding uncoupled ROS production. When paired with observations that P450-specific genotype/phenotype associations and the untold num- alternative splicing is a primary mechanism by which mitochondria bers of unclassified SNPs, it becomes clear why accurate genetic alter cellular phenotypes to mitigate environmental stress (Guantes screening remains challenging, and only one aspect of the therapeutic et al., 2015), the recognition of new, nonclassic roles for P450 variants decision-making process. linked to disease seems probable. Ultimately, the falling cost and broader availability of pyrosequencing In this regard, it is interesting that the consensus sequence (GU-AG) technologies support our call for improved RNA sequence strategies for of most RNA splice junctions contains a minimum of two guanine personalized medicine, as accurate identification of the patient’s functional nucleotides, which are highly sensitive to ROS-mediated oxidation. In genome is a crucial component of precision medicine. Although tran- the presence of radical oxygen, guanine is rapidly converted to 8-oxo- scriptomic analysis offers superior guidance in the design of personalized guanine and related metabolites that disrupt normal base pairing and local therapeutic options, its broad implementation will require technical secondary structure. Formation of 8-oxo-guanine at splice junctions can improvements to sample collection and processing that are also problem- impede target site recognition by splicing factors (e.g., snRNAs and atic for genomic testing. In this regard, complementary metabolomics Alternative P450 Splicing Redirects Human Drug Metabolism 385 approaches directed at variant-specific metabolism may provide more P450s like CYP3A4 (Arora and Iversen, 2001), whose metabolism of feasible, short-term improvements to PGx screening and precision-based drugs like tamoxifen is linked to genotoxicity (Mahadevan et al., 2006). As approaches to medicine (Beger et al., 2016). Innovative, gene-directed our appreciation for transcriptome expansion and the mechanisms of therapeutic technologies such as splice-altering antisense oligonucleotides alternative P450 gene-splicing evolve, new therapeutic gene-editing and CRISPR/Cas9 genome-editing systems may also become feasible options will probably emerge that could scarcely be predicted using tools for manipulating a patient’s transcriptome to optimize therapeutic genetic testing alone. outcomes. Key examples of splice-switching technology already being In summary, the human cytochrome P450 family transcriptome investigated to treat human disease are listed in Supplemental Table 2. In contains over 965 different variant forms (Table 2), many with common this regard, our group has participated in the development of eteplirsen, the structural features sensitive to alternative splicing events that expand new antisense oligonucleotide drug that received accelerated approval P450 protein diversity. The transcription and processing of P450 gene from the FDA for the treatment of Duchenne muscular dystrophy (DMD; transcripts is complex and coordinately regulated within the nucleus by Syed, 2016; Niks and Aartsma-Rus, 2017). Eteplirsen’s development multiple factors, including NR signaling via environmental sensors like evolved from early studies of exon skipping in the murine dystrophin the peroxisome proliferator–activated receptors (PPARs) PPARg and model (Fall et al., 2006; Fletcher et al., 2007; Adams et al., 2007; Mitrpant PPARa, which interact with the PGC-1a transcriptional coactivator to et al., 2009), a canine model of DMD (McClorey et al., 2006b), and in regulate oxidative metabolism and mitochondrial biogenesis (Wu et al., human muscle explants (McClorey et al., 2006a). Our group has also 1999; Monsalve et al., 2000). Multiple steroids, including products of employed exon-skipping oligomers to refine the immune response– CYPs 1A, 1B, 2A, 2B, 2C, 2D, 3A, 7A, 17A, 19A, 24A1, and 51A

mediated gene expression of CD45 protein-tyrosine phosphatase in a metabolism, bind NRs and lead to interactions with NR coregulators Downloaded from murine anthrax model (Panchal et al., 2009; Mourich and Iversen, 2009), through LxxLL or FxxLF motifs that modulate the assembly of the of interleukin-10 in an Ebola virus lethal-challenge mouse model (Panchal spliceosome complex and pre-mRNA splicing (Auboeuf et al., 2005). et al., 2014) and of CTLA-4 in a murine model of autoimmune diabetes The androgen receptor (AR), which binds multiple metabolites of CYPs (Mourich et al., 2014). We hypothesize that similar splice-altering 2A, 2C, 2D, 3A, 17A, 19A, and 21A, can also directly interact with technology may be useful in redirecting the function of drug metabolizing nucleolar splicing factors (e.g., U5 small nucleolar ribonuclear protein), dmd.aspetjournals.org at ASPET Journals on October 3, 2021

Fig. 4. Endoxenobiotic crosstalk among cytochrome P450 and nuclear receptor genes coordinate alternative splicing and resemble a primitive immune system. Human tissues are subject to exposure from over 400 FDA-approved drugs, .10,000 xenobiotics, and untold numbers of endogenous substrates and their metabolites (x . 100,000). Cytochrome P450 genes participate in phase I detoxification of many of these compounds, including model substrates benzo[a]pyrene (via CYP3A4) and calcifediol (via CYP24A1). P450 genes are classically induced to silence endoxenobiotic signaling through cognate nuclear receptors, which modulate global gene expression and splicing events by “coloring” or modulating the composition of coregulatory factors that comprise both the transcription complex and the spliceosome complex, which ultimately alter the nature of both ribosome assembly and gene expression (Auboeuf et al., 2005). Model substrates are subject to metabolism by a finite population of P450s in a given tissue, however, and because each gene is sensitive to an infinite number of environmentally sensitive, alternative splicing events, each individual may express a unique, tissue-specific “P450 gene cloud” comprising both wild-type (WT) and splice-altered variant forms (e.g., SV1, SV2, etc.). P450 splice variants can: 1) display reduced ability to metabolize model substrates, 2) function as dominant negatives to sequester compounds from metabolism or potentiate basal NR-mediated signaling, or 3) function as a conformationally distinct protein with alternative metabolic function or cellular role. When coupled with existing paradigms of alternate P450 trafficking and membrane- associated , an integrated network of crosstalk among 57 P450 and 48 NR genes begins to emerge, as novel P450 metabolites may engage NR signaling pathways in unique ways that reprogram gene splicing and expression to promote cellular homeostasis in the face of endocrine disruption. NR signaling cascades can alter both the transcriptome and epigenome of an individual, providing an elegant feedback mechanisms for adaptation to cellular stress created by unique personal history and disease status. 386 Annalora et al. indicating a receptor-mediated role in transcription that is coupled to pre- References mRNA splicing mechanisms (Zhao et al., 2002). Vitamin D receptor Abraham JE, Maranian MJ, Driver KE, Platte R, Kalmyrzaev B, Baynes C, Luccarini C, Shah M, activation mediated by metabolites of CYPs 2R, 2J, 3A, 11A, 27A, 24A, Ingle S, Greenberg D, et al. (2010) CYP2D6 gene variants: association with breast cancer specific survival in a cohort of breast cancer patients from the United Kingdom treated with and 27B can also alter P450 gene expression and splicing through adjuvant tamoxifen. Breast Cancer Res 12:R64–R75. NR-mediated crosstalk (e.g., PPARs) transduced via interactions with the Abreu MT, Kantorovich V, Vasiliauskas EA, Gruntmanis U, Matuk R, Daigle K, Chen S, Zehnder D, Lin YC, Yang H, et al. (2004) Measurement of vitamin D levels in inflammatory bowel retinoid X receptor (Matsuda and Kitagishi, 2013), recruitment of the disease patients reveals a subset of Crohn’s disease patients with elevated 1,25-dihydroxyvitamin NCoA62/SKIP coactivator complex (Zhang et al., 2003), and discrete D and low bone mineral density. Gut 53:1129–1136. interactions with the heterogeneous nuclear ribonucleoprotein C-splicing Adams AM, Harding PL, Iversen PL, Coleman C, Fletcher S, and Wilton SD (2007) Antisense oligonucleotide induced exon skipping and the dystrophin gene transcript: cocktails and factor (Zhou et al., 2015). Traditional VDR signaling in the nucleus is chemistries. BMC Mol Biol 8:57–65. further refined by several nontraditional NR functions operating near the Allorge D, Chevalier D, Lo-Guidice JM, Cauffiez C, Suard F, Baumann P, Eap CB, and Broly F (2003) Identification of a novel splice-site mutation in the CYP1A2 gene. Br J Clin Pharmacol plasma membrane that alter gene expression via modulation of key 56:341–344. membrane-based paracrine signaling pathways, mediated by agents like Anandatheerthavarada HK, Addya S, Dwivedi RS, Biswas G, Mullick J, and Avadhani NG (1997) Localization of multiple forms of inducible cytochromes P450 in rat liver mitochondria: im- Wnt and epidermal growth factor (Larriba et al., 2014). Vitamin A munological characteristics and patterns of xenobiotic substrate metabolism. Arch Biochem metabolites (or retinoids) of CYPs 1A, 2B, 2C, and 26, signaling through Biophys 339:136–150. Annalora A, Bobrovnikova-Marjon E, Serda R, Lansing L, Chiu ML, Pastuszyn A, Iyer S, Marcus the retinoic acid receptor and retinoid X receptor, are also known to guide CB, and Omdahl JL (2004) Rat cytochrome P450C24 (CYP24A1) and the role of F249 in the recruitment of SC35 coactivators to regulate the alternate splicing of substrate binding and catalytic activity. Arch Biochem Biophys 425:133–146. protein kinase delta (PKCd) among other pre-mRNAs (Apostolatos et al., Annalora AJ, Goodin DB, Hong WX, Zhang Q, Johnson EF, and Stout CD (2010) Crystal structure of CYP24A1, a mitochondrial cytochrome P450 involved in vitamin D metabolism. JMolBiol

2010). Collectively, these data reveal a novel P450-based mechanism for 396:441–451. Downloaded from adaptive transcriptome remodeling, whereby xenobiotics and endogenous Apostolatos H, Apostolatos A, Vickers T, Watson JE, Song S, Vale F, Cooper DR, Sanchez-Ramos “ J, and Patel NA (2010) Vitamin A metabolite, all-trans-retinoic acid, mediates alternative substrates, monitored by one of several tissue- or disease-specific P450 splicing of protein kinase C deltaVIII (PKCdeltaVIII) isoform via splicing factor SC35. J Biol clouds,” are metabolized in a coordinated fashion that harmonize NR Chem 285:25987–25995. Ariyoshi N, Shimizu Y, Kobayashi Y, Nakamura H, Nakasa H, Nakazawa K, Ishii I, and Kitada M signaling cascades with alternative gene expression and splicing events (2007) Identification and partial characterization of a novel CYP2C9 splicing variant encoding a that promote adaptive responses to cell stress or stimuli (Fig. 4). protein lacking eight amino acid residues. Drug Metab Pharmacokinet 22:187–194. Arora V and Iversen PL (2001) Redirection of drug metabolism using antisense technology. Curr

In conclusion, the human metabolome adapts to substrate burden dmd.aspetjournals.org Opin Mol Ther 3:249–257. through the induction of gene transcription, which helps to maintain Auboeuf D, Dowhan DH, Dutertre M, Martin N, Berget SM, and O’Malley BW (2005) A subset of homeostasis in a well documented pathway guided by NR binding and nuclear receptor coregulators act as coupling proteins during synthesis and maturation of RNA transcripts. Mol Cell Biol 25:5307–5316. signaling events. In this respect, the metabolic response to xenobiotics Bailey D, Veljkovic K, Yazdanpanah M, and Adeli K (2013) Analytical measurement and clinical (via P450 induction) is adaptive in a manner reminiscent of the immune relevance of vitamin D(3) C3-epimer. Clin Biochem 46:190–196. Bajpai P, Srinivasan S, Ghosh J, Nagy LD, Wei S, Guengerich FP, and Avadhani NG (2014) Targeting response to viral antigen; there is a recognition phase of the chemical by of splice variants of human cytochrome P450 2C8 (CYP2C8) to mitochondria and their role in the P450 active site, an activation phase when the chemical (or P450 metabolism and respiratory dysfunction. JBiolChem289:29614 –29630. Bauer M, Herbarth O, Aust G, and Graebsch C (2005) Molecular cloning and expression of novel metabolite) interacts with the NR, and an effector phase in which the alternatively spliced cytochrome P450 2E1 mRNAs in humans. Mol Cell Biochem 280:201–207. at ASPET Journals on October 3, 2021 coordinated transcription and splicing of P450 transcripts occurs to Bauer M, Herbarth O, Rudzok S, Schmücking E, Müller A, Aust G, and Gräbsch C (2007) feedback-modulate NR signaling. The analogy to the immune response Diversity of common alternative splicing variants of human cytochrome P450 1A1 and their association to carcinogenesis. Int J Oncol 31:211–218. is appropriate here in that specific transcript variants are produced in Beger RD, Dunn W, Schmidt MA, Gross SS, Kirwan JA, Cascante M, Brennan L, Wishart DS, response to a specific chemical stimulus. The ability to tightly control Oresic M, Hankemeier T et al. for “Precision Medicine and Pharmacometabolomics Task Group”—Metabolomics Society Initiative (2016) Metabolomics enables precision medicine: “a cellular homeostasis via NR-mediated gene expression and alternative white paper, community perspective”. Metabolomics 12:149. splicing implies a high order of sophistication operating in what might be Black DL (2000) Protein diversity from alternative splicing: a challenge for bioinformatics and post-genome biology. Cell 103:367–370. considered a primitive, chemical immune response. Molecules that engage Blencowe BJ (2006) Alternative splicing: new insights from global analyses. Cell 126:37–47. this primitive P450-based immune system transduce transcriptome-wide Bouraïma H, Hanoux V, Mittre H, Féral C, Benhaïm A, and Leymarie P (2001) Expression of the rabbit cytochrome P450 aromatase encoding gene uses alternative tissue-specific promoters. Eur biologic responses capable of reshaping both the phenotype and J Biochem 268:4506–4512. “epigenotype” of the cell (via the regulation of both coding and Brändén G, Sjögren T, Schnecke V, and Xue Y (2014) Structure-based ligand design to overcome CYP inhibition in drug discovery projects. Drug Discov Today 19:905–911. noncoding RNA), allowing for reversible environmental adaptation, as Busi F and Cresteil T (2005) CYP3A5 mRNA degradation by nonsense-mediated mRNA decay. well as imprinting, which in rare cases may persist transgenerationally Mol Pharmacol 68:808–815. Carr DF, Alfirevic A, and Pirmohamed M (2014) Pharmacogenomics: Current State-of-the-Art. (Hochberg et al., 2011). Improved knowledge of both the adaptive and Genes (Basel) 5:430–443. maladaptive epigenome remodeling processes induced by xenobiotics may Caudle KE, Gammal RS, Whirl-Carrillo M, Hoffman JM, Relling MV, and Klein TE (2016) Evidence and resources to implement pharmacogenetic knowledge for precision medicine. Am J ultimately help reconcile interindividual variability in efficacy and toxicity Health Syst Pharm 73:1977–1985. that plague many FDA-approved drugs. These insights will provide new Chen PH, Lee KW, Hsu CC, Chen JY, Wang YH, Chen KK, Wang HM, Huang HW, and Huang B guidance for developing “individualized” therapeutic strategies more (2014) Expression of a splice variant of CYP26B1 in betel quid-related oral cancer. Sci World J 2014:810561. sensitive to a patient’s adaptive transcriptome or functional genome, which Chen W, Kubota S, Ujike H, Ishihara T, and Seyama Y (1998) A novel Arg362Ser mutation in the as exemplified by P450 superfamily of genes is the ultimate expression of sterol 27-hydroxylase gene (CYP27): its effects on pre-mRNA splicing and enzyme activity. Biochemistry 37:15050–15056. the heritable epigenotype and appears to remain environmentally Chinta SJ, Kommaddi RP, Turman CM, Strobel HW, and Ravindranath V (2005) Constitutive responsive throughout all phases of the human life cycle. expression and localization of cytochrome P-450 1A1 in rat and human brain: presence of a splice variant form in human brain. J Neurochem 93:724–736. Christmas P, Jones JP, Patten CJ, Rock DA, Zheng Y, Cheng SM, Weber BM, Carlesso N, Scadden DT, Rettie AE, et al. (2001) Alternative splicing determines the function of CYP4F3 by Acknowledgments switching substrate specificity. J Biol Chem 276 :38166–38172. The authors thank Dr. Ronald N. Hines (US-EPA) for his thoughtful comments Christmas P, Ursino SR, Fox JW, and Soberman RJ (1999) Expression of the CYP4F3 gene. tissue- specific splicing and alternative promoters generate high and low K(m) forms of leukotriene B(4) and suggestions concerning this review. omega-hydroxylase. J Biol Chem 274:21191–21199. Costa-Santos M, Kater CE, Dias EP, and Auchus RJ (2004) Two intronic mutations cause Authorship Contributions 17-hydroxylase deficiency by disrupting splice acceptor sites: direct demonstration of aberrant splicing and absent enzyme activity by expression of the entire CYP17 gene in HEK-293 cells. J Participated in research design: Annalora, Iversen, Marcus. Clin Endocrinol Metab 89:43–48. Performed data analysis: Annalora, Iversen. Cowie P, Hay EA, and MacKenzie A (2015) The noncoding human genome and the future of personalised medicine. Expert Rev Mol Med 17:e4. Wrote or contributed to the writing of the manuscript: Annalora, Iversen, Cunningham F, Amode MR, Barrell D, Beal K, Billis K, Brent S, Carvalho-Silva D, Clapham P, Marcus. Coates G, Fitzgerald S, et al. (2015) Ensembl 2015. Nucleic Acids Res 43:D662–D669. Alternative P450 Splicing Redirects Human Drug Metabolism 387

Crews KR, Gaedigk A, Dunnenberger HM, Leeder JS, Klein TE, Caudle KE, Haidar CE, Shen DD, Huang Z, Fasco MJ, Figge HL, Keyomarsi K, and Kaminsky LS (1996) Expression of cytochromes Callaghan JT, Sadhasivam S, et al.; Clinical Pharmacogenetics Implementation Consortium P450 in human breast tissue and tumors. Drug Metab Dispos 24:899–905. (2014) Clinical Pharmacogenetics Implementation Consortium guidelines for cytochrome P450 Huang Z, Fasco MJ, Spivack S, and Kaminsky LS (1997) Comparisons of CYP2D messenger 2D6 genotype and codeine therapy: 2014 update. Clin Pharmacol Ther 95:376–382. RNA splice variant profiles in human lung tumors and normal tissues. Cancer Res 57: de Morais SMF, Wilkinson GR, Blaisdell J, Nakamura K, Meyer UA, and Goldstein JA (1994) The 2589–2592. major genetic defect responsible for the polymorphism of S- metabolism in humans. Hwang DY, Hung CC, Riepe FG, Auchus RJ, Kulle AE, Holterhus PM, Chao MC, Kuo MC, J Biol Chem 269:15419–15422. Hwang SJ, and Chen HC (2011) CYP17A1 intron mutation causing cryptic splicing in 17a- Denson J, Wu Y, Yang W, and Zhang J (2005) Inter-individual variation of several cytochrome hydroxylase deficiency. PLoS One 6:e25492. P450 2D6 splice variants in human liver. Biochem Biophys Res Commun 330:498–504. Ibeanu GC, Blaisdell J, Ferguson RJ, Ghanayem BI, Brosen K, Benhamou S, Bouchardy C, Desrochers M, Christou M, Jefcoate C, Belzil A, and Anderson A (1996) New proteins in the rat Wilkinson GR, Dayer P, and Goldstein JA (1999) A novel transversion in the intron 5 donor CYP2B subfamily: presence in liver microsomes of the constitutive CYP2B3 protein and the splice junction of CYP2C19 and a sequence polymorphism in exon 3 contribute to the poor -inducible protein product of alternatively spliced CYP2B2 mRNA. Biochem metabolizer phenotype for the drug S-mephenytoin. JPharmacolExpTher290: Pharmacol 52:1311–1319. 635–640. Diesel B, Seifert M, Radermacher J, Fischer U, Tilgen W, Reichrath J, and Meese E (2004) Johnson CS, Chung I, and Trump DL (2010) Epigenetic silencing of CYP24 in the tumor mi- Towards a complete picture of splice variants of the gene for 25-hydroxyvitamin D31a- croenvironment. J Steroid Biochem Mol Biol 121:338–342. hydroxylase in brain and skin cancer. J Steroid Biochem Mol Biol 89-90:527–532. Johnson EF and Stout CD (2013) Structural diversity of eukaryotic membrane cytochrome p450s. J Ding S, Lake BG, Friedberg T, and Wolf CR (1995) Expression and alternative splicing of the Biol Chem 288:17082–17090. cytochrome P-450 CYP2A7. Biochem J 306:161–166. Kimouli M, Gourvas V, Konstantoudaki X, Basta M, Miyakis S, and Spandidos DA (2009) The Doleschall M, Szabó JA, Pázmándi J, Szilágyi Á, Koncz K, Farkas H, Tóth M, Igaz P, Gláz E, effect of an exon 12 polymorphism of the human thromboxane synthase (CYP5A1) gene in Prohászka Z, et al. (2014) Common genetic variants of the human steroid 21-hydroxylase gene stroke patients. Med Sci Monit 15:BR30–BR35. (CYP21A2) are related to differences in circulating hormone levels. PLoS One 9:e107244. Knüpfer H, Schmidt R, Stanitz D, Brauckhoff M, Schönfelder M, and Preiss R (2004) CYP2C and Dong H, Shertzer HG, Genter MB, Gonzalez FJ, Vasiliou V, Jefcoate C, and Nebert DW (2013) IL-6 expression in breast cancer. Breast 13:28–34. Mitochondrial targeting of mouse NQO1 and CYP1B1 proteins. Biochem Biophys Res Commun Kommaddi RP, Turman CM, Moorthy B, Wang L, Strobel HW, and Ravindranath V (2007) An 435:727–732. alternatively spliced cytochrome P4501A1 in human brain fails to bioactivate polycyclic aro- Elens L, Bouamar R, Hesselink DA, Haufroid V, van der Heiden IP, van Gelder T, and van Schaik matic hydrocarbons to DNA-reactive metabolites. J Neurochem 102:867–877. RH (2011) A new functional CYP3A4 intron 6 polymorphism significantly affects tacrolimus Kramlinger VM, Nagy LD, Fujiwara R, Johnson KM, Phan TT, Xiao Y, Enright JM, Toomey MB, Downloaded from pharmacokinetics in kidney transplant recipients. Clin Chem 57:1574–1583. Corbo JC, and Guengerich FP (2016) Human cytochrome P450 27C1 catalyzes 3,4-desaturation Eliasson E and Kenna JG (1996) Cytochrome P450 2E1 is a cell surface autoantigen in halothane of retinoids. FEBS Lett 590:1304–1312. hepatitis. Mol Pharmacol 50:573–582. Kristensen VN, Harada N, Yoshimura N, Haraldsen E, Lonning PE, Erikstein B, Kåresen R, Elmabsout AA, Kumawat A, Saenz-Méndez P, Krivospitskaya O, Sävenstrand H, Olofsson PS, Kristensen T, and Børresen-Dale AL (2000) Genetic variants of CYP19 (aromatase) and breast Eriksson LA, Strid A, Valen G, Törmä H, et al. (2012) Cloning and functional studies of a splice cancer risk. Oncogene 19:1329–1333. variant of CYP26B1 expressed in vascular cells. PLoS One 7:e36839. Kuehl P, Zhang J, Lin Y, Lamba J, Assem M, Schuetz J, Watkins PB, Daly A, Wrighton SA, Hall Erratums. Nature 2001; 411 (6838):720; Nature 2001; 412 (6846):565. SD, et al. (2001) Sequence diversity in CYP3A promoters and characterization of the genetic Faiq MA, Dada R, Sharma R, Saluja D, and Dada T (2014) CYP1B1: a unique gene with unique basis of polymorphic CYP3A5 expression. Nat Genet 27:383–391. characteristics. Curr Drug Metab 15:893–914. La Cognata V, Iemmolo R, D’Agata V, Scuderi S, Drago F, Zappia M, and Cavallaro S (2014) dmd.aspetjournals.org Fall AM, Johnsen R, Honeyman K, Iversen P, Fletcher S, and Wilton SD (2006) Induction of Increasing the coding potential of genomes through alternative splicing: The case of PARK2 revertant fibres in the mdx mouse using antisense oligonucleotides. Genet Vaccines Ther 4:3–15. gene. Curr Genomics 15:203–216. Finta C and Zaphiropoulos PG (2000) The human cytochrome P450 3A locus. Gene evolution by Lamb DC and Waterman MR (2013) Unusual properties of the cytochrome P450 superfamily. capture of downstream exons. Gene 260:13–23. Philos Trans R Soc Lond B Biol Sci 368:20120434. Finta C and Zaphiropoulos PG (2002) Intergenic mRNA molecules resulting from trans-splicing. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle J Biol Chem 277:5882–5890. M, FitzHugh W, et al.; International Human Genome Sequencing Consortium (2001) Initial Fischer D, Becker S, Cordes T, Bücker B, Diedrich K, Friedrich M, Salehin D, and Thill M (2009a) sequencing and analysis of the human genome. Nature 409:860–921. Vitamin D-24-hydroxylase in benign and malignant breast tissue and cell lines. Anticancer Res Larriba MJ, González-Sancho JM, Bonilla F, and Muñoz A (2014) Interaction of vitamin D with 29:3641–3645. membrane-based signaling pathways. Front Physiol 5:60. Fischer D, Seifert M, Becker S, Ludders D, Cordes T, Reichrath J, and Friedrich M (2007) 25- Lee HH (2013) Variants of the CYP21A2 and CYP21A1P genes in congenital adrenal hyperplasia. at ASPET Journals on October 3, 2021 Hydroxyvitamin D3 1alpha-hydroxylase splice variants in breast cell lines MCF-7 and MCF-10. Clin Chim Acta 418:37–44. Cancer Genomics Proteomics 4:295–300. Lee SJ, van der Heiden IP, Goldstein JA, and van Schaik RH (2007) A new CYP3A5 variant, Fischer D, Thomé M, Becker S, Cordes T, Diedrich K, Friedrich M, and Thill M (2009b) 25- CYP3A5*11, is shown to be defective in nifedipine metabolism in a recombinant cDNA ex- Hydroxyvitamin D3 1alpha-hydroxylase splice variants in benign and malignant ovarian cell pression system. Drug Metab Dispos 35:67–71. lines and tissue. Anticancer Res 29:3627–3633. Lejeune F and Maquat LE (2005) Mechanistic links between nonsense-mediated mRNA decay and Flanagan JN, Wang L, Tangpricha V, Reichrath J, Chen TC, and Holick MF (2003) Regulation of pre-mRNA splicing in mammalian cells. Curr Opin Cell Biol 17:309–315. the 25-hydroxyvitamin D-1alpha-hydroxylase gene and its splice variant. Recent Results Cancer Leung YK, Lau KM, Mobley J, Jiang Z, and Ho SM (2005) Overexpression of cytochrome P450 Res 164:157–167. 1A1 and its novel spliced variant in ovarian cancer cells: alternative subcellular enzyme com- Fletcher S, Honeyman K, Fall AM, Harding PL, Johnsen RD, Steinhaus JP, Moulton HM, Iversen partmentation may contribute to carcinogenesis. Cancer Res 65:3726–3734. PL, and Wilton SD (2007) Morpholino oligomer-mediated exon skipping averts the onset of Lewiñska M, Zmrzljak UP, and Rozman D (2013) Low nucleotide variability of CYP51A1 in dystrophic pathology in the mdx mouse. Mol Ther 15:1587–1592. humans: meta-analysis of cholesterol and bile acid synthesis and xenobiotic metabolism path- García-Roca P, Medeiros M, Reyes H, Rodríguez-Espino BA, Alberú J, Ortiz L, Vásquez-Perdomo ways. Acta Chim Slov 60:875–883. M, Elizondo G, Morales-Buenrostro LE, Mancilla Urrea E, et al. (2012) CYP3A5 polymorphism Li F, Jiang C, Larsen MC, Bushkofsky J, Krausz KW, Wang T, Jefcoate CR, and Gonzalez FJ in Mexican renal transplant recipients and its association with tacrolimus dosing. Arch Med Res (2014) Lipidomics reveals a link between CYP1B1 and SCD1 in promoting obesity. JProteome 43:283–287. Res 13:2679–2687. Garuti R, Lelli N, Barozzini M, Tiozzo R, Dotti MT, Federico A, Ottomano AM, Croce A, Li M, Wang W, Li Y, Wang L, Shen X, and Tang Z (2013a) CYP46A1 intron-2T/C polymorphism Bertolini S, and Calandra S (1996) Cerebrotendinous xanthomatosis caused by two new mu- and Alzheimer ’s disease: an updated meta-analysis of 16 studies including 3,960 cases and 3,828 tations of the sterol-27-hydroxylase gene that disrupt mRNA splicing. JLipidRes37:1459–1467. controls. Neurosci Lett 549:18–23. Ghosh D, Lo J, Morton D, Valette D, Xi J, Griswold J, Hubbell S, Egbuta C, Jiang W, An J, et al. Li L, Yin Z, Liu J, Li G, Wang Y, Yan J, and Zhou H (2013b) CYP46A1 T/C polymorphism (2012) Novel aromatase inhibitors by structure-guided design. J Med Chem 55:8464–8476. associated with the APOE «4 allele increases the risk of Alzheimer’s disease. JNeurol260: Gomez A, Nekvindova J, Travica S, Lee MY, Johansson I, Edler D, Mkrtchian S, and Ingelman- 1701–1708. Sundberg M (2010) Colorectal cancer-specific cytochrome P450 2W1: intracellular localization, Lin L, Ercan O, Raza J, Burren CP, Creighton SM, Auchus RJ, Dattani MT, and Achermann JC glycosylation, and catalytic activity. Mol Pharmacol 78:1004–1011. (2007) Variable phenotypes associated with aromatase (CYP19) insufficiency in humans. JClin Gonzàlez-Porta M, Frankish A, Rung J, Harrow J, and Brazma A (2013) Transcriptome analysis of Endocrinol Metab 92:982–990. human tissues and cell lines reveals one dominant transcript per gene. Genome Biol 14:R70. Lin YS, Dowling ALS, Quigley SD, Farin FM, Zhang J, Lamba J, Schuetz EG, and Thummel KE Guantes R, Rastrojo A, Neves R, Lima A, Aguado B, and Iborra FJ (2015) Global variability in gene (2002) Co-regulation of CYP3A4 and CYP3A5 and contribution to hepatic and intestinal expression and alternative splicing is modulated by mitochondrial content. Genome Res 25:633–644. midazolam metabolism. Mol Pharmacol 62:162–172. Guengerich FP (2013) New trends in cytochrome p450 research at the half-century mark. J Biol Liu L, Bai YX, Zhou JH, Sun XW, Sui H, Zhang WJ, Yuan HH, Xie R, Wei XL, Zhang TT, et al. Chem 288:17063–17064. (2013a) A polymorphism at the 39-UTR region of the aromatase gene is associated with the Hadfield KD and Newman WG (2012) Pharmacogenetics of aromatase inhibitors. Pharmacoge- efficacy of the aromatase inhibitor, , in metastatic breast carcinoma. Int J Mol Sci 14: nomics 13:699–707. 18973–18988. Hampf M, Dao NT, Hoan NT, and Bernhardt R (2001) Unequal crossing-over between aldosterone Liu X, Low SK, and Boddy AV (2016) The implications of genetic variation for the pharmaco- synthase and 11beta-hydroxylase genes causes congenital adrenal hyperplasia. J Clin Endocrinol kinetics and pharmacodynamics of aromatase inhibitors. Expert Opin Drug Metab Toxicol 12: Metab 86:4445 –4452. 851–863. Hanahan D and Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674. Liu YL, Xu Y, Li F, Chen H, and Guo SL (2013b) CYP2A6 deletion polymorphism is associated Hecker M and Ullrich V (1989) On the mechanism of prostacyclin and thromboxane A2 bio- with decreased susceptibility of lung cancer in Asian smokers: a meta-analysis. Tumour Biol 34: synthesis. J Biol Chem 264:141–150. 2651–2657. Hines RN and McCarver DG (2002) The ontogeny of human drug-metabolizing enzymes: phase I Lu Y, Mo C, Zeng Z, Chen S, Xie Y, Peng Q, He Y, Deng Y, Wang J, Xie L, et al. (2013) oxidative enzymes. J Pharmacol Exp Ther 300:355–360. CYP2D6*4 allele polymorphism increases the risk of Parkinson’s disease: evidence from meta- Hochberg Z, Feil R, Constancia M, Fraga M, Junien C, Carel JC, Boileau P, Le Bouc Y, Deal CL, analysis. PLoS One 8:e84413. Lillycrop K, et al. (2011) Child health, developmental plasticity, and epigenetic programming. Lum DW, Perel P, Hingorani AD, and Holmes MV (2013) CYP2D6 genotype and tamoxifen Endocr Rev 32:159–224. response for breast cancer: a systematic review and meta-analysis. PLoS One 8:e76648. Horváth HC, Khabir Z, Nittke T, Gruber S, Speer G, Manhardt T, Bonner E, and Kallay E (2010) Maas RM, Reus K, Diesel B, Steudel WI, Feiden W, Fischer U, and Meese E (2001) Amplification CYP24A1 splice variants—implications for the antitumorigenic actions of 1,25-(OH)2D3 in and expression of splice variants of the gene encoding the P450 cytochrome 25-hydroxyvitamin colorectal cancer. J Steroid Biochem Mol Biol 121:76–79. D(3) 1,alpha-hydroxylase (CYP 27B1) in human malignant glioma. Clin Cancer Res 7:868–875. 388 Annalora et al.

Mahadevan B, Arora V, Schild LJ, Keshava C, Cate ML, Iversen PL, Poirier MC, Weston A, polymorphism of the human retinoic acid-metabolizing enzyme CYP26A1, an enzyme that may Pereira C, and Baird WM (2006) Reduction in tamoxifen-induced CYP3A2 expression and be involved in spina bifida. Birth Defects Res A Clin Mol Teratol 76:491–498. DNA adducts using antisense technology. Mol Carcinog 45:118–125. Ren S, Nguyen L, Wu S, Encinas C, Adams JS, and Hewison M (2005) Alternative splicing of Mangin M, Sinha R, and Fincher K (2014) and vitamin D: the infection connection. vitamin D-24-hydroxylase: a novel mechanism for the regulation of extrarenal 1,25-dihydrox- Inflamm Res 63:803–819. yvitamin D synthesis. J Biol Chem 280:20604–20611. Matsuda S and Kitagishi Y (2013) Peroxisome proliferator-activated receptor and vitamin d re- Rhieu SY, Annalora AJ, Wang G, Flarakos CC, Gathungu RM, Vouros P, Sigüeiro R, Mouriño A, ceptor signaling pathways in cancer cells. Cancers (Basel) 5:1261–1270. Schuster I, Palmore GT, et al. (2013) Metabolic stability of 3-epi-1a,25-dihydroxyvitamin D3 McClorey G, Fall AM, Moulton HM, Iversen PL, Rasko JE, Ryan M, Fletcher S, and Wilton SD over 1 a 25-dihydroxyvitamin D3: metabolism and molecular docking studies using rat (2006a) Induced dystrophin exon skipping in human muscle explants. Neuromuscul Disord 16: CYP24A1. J Cell Biochem 114:2293–2305. 583–590. Robins T, Carlsson J, Sunnerhagen M, Wedell A, and Persson B (2006) Molecular model of human McClorey G, Moulton HM, Iversen PL, Fletcher S, and Wilton SD (2006b) Antisense CYP21 based on mammalian CYP2C5: structural features correlate with clinical severity of oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model mutations causing congenital adrenal hyperplasia. Mol Endocrinol 20:2946–2964. of DMD. Gene Ther 13:1373–1381. Rogozin IB, Carmel L, Csuros M, and Koonin EV (2012) Origin and evolution of spliceosomal McGlincy NJ and Smith CW (2008) Alternative splicing resulting in nonsense-mediated mRNA introns. Biol Direct 7:11. decay: what is the meaning of nonsense? Trends Biochem Sci 33:385–393. Roy B, Haupt LM, and Griffiths LR (2013) Review: Alternate splicing (AS) of genes as an Meyer UA (2007) Endo-xenobiotic crosstalk and the regulation of cytochromes P450. Drug Metab approach for generating protein complexity. Curr Genomics 14:182–194. Rev 39:639–646. Salzman J, Gawad C, Wang PL, Lacayo N, and Brown PO (2012) Circular RNAs are the pre- Miles JS, McLaren AW, and Wolf CR (1989) Alternative splicing in the human cytochrome dominant transcript isoform from hundreds of human genes in diverse cell types. PLoS One 7: P450IIB6 gene generates a high level of aberrant messages. Nucleic Acids Res 17:8241–8255. e30733. Mitrpant C, Fletcher S, Iversen PL, and Wilton SD (2009) By-passing the nonsense mutation in the Sangar MC, Anandatheerthavarada HK, Martin MV, Guengerich FP, and Avadhani NG (2010) 4 CV mouse model of muscular dystrophy by induced exon skipping. JGeneMed11:46–56. Identification of genetic variants of human cytochrome P450 2D6 with impaired mitochondrial Monsalve M, Wu Z, Adelmant G, Puigserver P, Fan M, and Spiegelman BM (2000) Direct targeting. Mol Genet Metab 99:90–97. coupling of transcription and mRNA processing through the thermogenic coactivator PGC-1. Sarasquete ME, García-Sanz R, Marín L, Alcoceba M, Chillón MC, Balanzategui A, Santamaria C, Mol Cell 6:307–316. Rosiñol L, de la Rubia J, Hernandez MT, et al. (2008) Bisphosphonate-related osteonecrosis of Mort M, Ivanov D, Cooper DN, and Chuzhanova NA (2008) A meta-analysis of nonsense mu- the jaw is associated with polymorphisms of the cytochrome P450 CYP2C8 in multiple mye- tations causing human genetic disease. Hum Mutat 29:1037–1047. loma: a genome-wide single nucleotide polymorphism analysis. Blood 112:2709–2712. Downloaded from Mourich DV and Iversen PL (2009) Splicing in the immune system: potential targets for therapeutic Satyanarayana CR, Devendran A, Jayaraman M, Mannu J, Mathur PP, Gopal SD, Rajagopal K, intervention by antisense-mediated alternative splicing. Curr Opin Mol Ther 11:124–132. and Chandrasekaran A (2009) Influence of the genetic polymorphisms in the 59 flanking and Mourich DV, Oda SK, Schnell FJ, Crumley SL, Hauck LL, Moentenich CA, Marshall NB, exonic regions of CYP2C19 on proguanil oxidation. Drug Metab Pharmacokinet 24:537–548. Hinrichs DJ, and Iversen PL (2014) Alternative splice forms of CTLA-4 induced by antisense Scheible C, Thill M, Baum S, Solomayer E, and Friedrich M (2014) Implication of CYP24A1 mediated splice-switching influences autoimmune diabetes susceptibility in NOD mice. Nucleic splicing in breast cancer. Anticancer Agents Med Chem 14:109–114. Acid Ther 24:114–126. Searles Nielsen S, Bammler TK, Gallagher LG, Farin FM, Longstreth, JrW, Franklin GM, Swanson Muindi JR, Nganga A, Engler KL, Coignet LJ, Johnson CS, and Trump DL (2007) CYP24 splicing PD, and Checkoway H (2013) Genotype and age at Parkinson disease diagnosis. Int J Mol variants are associated with different patterns of constitutive and calcitriol-inducible CYP24 Epidemiol Genet 4:61–69. activity in human prostate cancer cell lines. J Steroid Biochem Mol Biol 103:334–337. Seifert M, Tilgen W, and Reichrath J (2009) Expression of 25-hydroxyvitamin D-1alpha-hydroxylase dmd.aspetjournals.org Nakayama T, Soma M, Saito S, Honye J, Yajima J, Rahmutula D, Kaneko Y, Sato M, Uwabo J, (1alphaOHase, CYP27B1) splice variants in HaCaT keratinocytes and other skin cells: modulation Aoi N, et al. (2002a) Association of a novel single nucleotide polymorphism of the prostacyclin by culture conditions and UV-B treatment in vitro. Anticancer Res 29:3659–3667. synthase gene with myocardial infarction. Am Heart J 143:797–801. Sevrioukova IF and Poulos TL (2013) Dissecting cytochrome P450 3A4-ligand interactions using Nakayama T, Soma M, Watanabe Y, Hasimu B, Sato M, Aoi N, Kosuge K, Kanmatsuse K, ritonavir analogues. Biochemistry 52:4474–4481. Kokubun S, Marrow JD, et al. (2002b) Splicing mutation of the gene in a Sharaf S, Hafez M, ElAbd D, Ismail A, Thabet G, and Elsharkawy M (2015) High frequency of family associated with hypertension. Biochem Biophys Res Commun 297:1135–1139. splice site mutation in 21-hydroxylase deficiency children. J Endocrinol Invest 38:505–511. Nelson DR (2003) Comparison of P450s from human and fugu: 420 million years of vertebrate Sheikh SA, Waryah AM, Narsani AK, Shaikh H, Gilal IA, Shah K, Qasim M, Memon AI, P450 evolution. Arch Biochem Biophys 409:18–24. Kewalramani P, and Shaikh N (2014) Mutational spectrum of the CYP1B1 gene in Pakistani Nelson DR, Zeldin DC, Hoffman SM, Maltais LJ, Wain HM, and Nebert DW (2004) Comparison patients with primary congenital glaucoma: novel variants and genotype-phenotype correlations. of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature Mol Vis 20:991–1001. at ASPET Journals on October 3, 2021 recommendations for genes, pseudogenes and alternative-splice variants. Pharmacogenetics 14: Shuldiner AR, O’Connell JR, Bliden KP, Gandhi A, Ryan K, Horenstein RB, Damcott CM, Pakyz 1–18. R, Tantry US, Gibson Q, et al. (2009) Association of cytochrome P450 2C19 genotype with the Nhamburo PT, Kimura S, McBride OW, Kozak CA, Gelboin HV, and Gonzalez FJ (1990) The antiplatelet effect and clinical efficacy of clopidogrel therapy. JAMA 302:849–857. human CYP2F gene subfamily: identification of a cDNA encoding a new cytochrome P450, Slavotinek AM, Mehrotra P, Nazarenko I, Tang PL, Lao R, Cameron D, Li B, Chu C, Chou C, cDNA-directed expression, and chromosome mapping. Biochemistry 29:5491–5499. Marqueling AL, et al. (2013) Focal facial dermal dysplasia, type IV, is caused by mutations in Niks EH and Aartsma-Rus A (2017) Exon skipping: a first in class strategy for Duchenne muscular CYP26C1. Hum Mol Genet 22:696–703. dystrophy. Expert Opin Biol Ther 17:225–236. Solomon O, Oren S, Safran M, Deshet-Unger N, Akiva P, Jacob-Hirsch J, Cesarkas K, Kabesa R, Ohgiya S, Komori M, Ohi H, Shiramatsu K, Shinriki N, and Kamataki T (1992) Six-base deletion Amariglio N, Unger R, et al. (2013) Global regulation of alternative splicing by adenosine occurring in messages of human cytochrome P-450 in the CYP2C subfamily results in reduction deaminase acting on RNA (ADAR). RNA 19:591–604. of tolbutamide hydroxylase activity. Biochem Int 27:1073–1081. Stelzer G, Dalah I, Stein TI, Satanower Y, Rosen N, Nativ N, Oz-Levi D, Olender T, Belinky F, Okino ST, Quattrochi LC, Pendurthi UR, McBride OW, and Tukey RH (1987) Characterization of Bahir I, et al. (2011) In-silico human genomics with GeneCards. Hum Genomics 5:709–717. multiple human cytochrome P-450 1 cDNAs. The chromosomal localization of the gene and Stenstedt K, Hallstrom M, Johansson I, Ingelman-Sundberg M, Ragnhammar P, and Edler D evidence for alternate RNA splicing. J Biol Chem 262:16072–16079. (2012) The expression of CYP2W1: a prognostic marker in colon cancer. Anticancer Res 32: Pagliarini V, Naro C, and Sette C (2015) Splicing Regulation: A Molecular Device to Enhance 3869–3874. Cancer Cell Adaptation. BioMed Res Int 2015:543067. St. Laurent G, Vyatkin Y, and Kapranov P (2014) Dark matter RNA illuminates the puzzle of Panchal RG, Ulrich RL, Bradfute SB, Lane D, Ruthel G, Kenny TA, Iversen PL, Anderson AO, genome-wide association studies. BMC Med 12:97–105. Gussio R, Raschke WC, et al. (2009) Reduced expression of CD45 protein-tyrosine phosphatase Stoilov I, Akarsu AN, Alozie I, Child A, Barsoum-Homsy M, Turacli ME, Or M, Lewis RA, provides protection against anthrax pathogenesis. J Biol Chem 284:12874–12885. Ozdemir N, Brice G, et al. (1998) Sequence analysis and homology modeling suggest that Panchal RG, Mourich DV, Bradfute S, Hauck LL, Warfield KL, Iversen PL, and Bavari S (2014) primary congenital glaucoma on 2p21 results from mutations disrupting either the hinge region Induced IL-10 splice altering approach to antiviral drug discovery. Nucleic Acid Ther 24:179–185. or the conserved core structures of cytochrome P4501B1. Am J Hum Genet 62:573–584. Park JW, Reed JR, and Backes WL (2015) The localization of cytochrome P450s CYP1A1 and Sun W, Li Y, Li J, Zhang Z, Zhu W, Liu W, Cai Q, Wang X, Cao L, Bai W, et al. (2015) Variant CYP1A2 into different lipid microdomains is governed by their N-terminal and internal protein recurrent risk among stroke patients with different CYP2C19 phenotypes and treated with clo- regions. J Biol Chem 290:29449–29460. pidogrel. Platelets 26:558–562. Peng X, Tiwari N, Roy S, Yuan L, Murillo G, Mehta RR, Benya RV, and Mehta RG (2012) Syed YY (2016) Eteplirsen: First Global Approval. Drugs 76:1699–1704. Regulation of CYP24 splicing by 1,25-dihydroxyvitamin D3 in human colon cancer cells. J Szabó JA, Szilágyi Á, Doleschall Z, Patócs A, Farkas H, Prohászka Z, Rácz K, Füst G, Endocrinol 212:207–215. and Doleschall M (2013) Both positive and negative selection pressures contribute to the Pepe CM, Saraco NI, Baquedano MS, Guercio G, Vaiani E, Marino R, Pandey AV, Flück CE, polymorphism pattern of the duplicated human CYP21A2 gene. PLoS One 8:e81977. Rivarola MA, and Belgorosky A (2007) The cytochrome P450 aromatase lacking exon 5 is Tanwar M, Dada T, Sihota R, Das TK, Yadav U, and Dada R (2009) Mutation spectrum of associated with a phenotype of nonclassic aromatase deficiency and is also present in normal CYP1B1 in North Indian congenital glaucoma patients. Mol Vis 15:1200–1209. human steroidogenic tissues. Clin Endocrinol (Oxf) 67:698–705. Thierry-Mieg D and Thierry-Mieg J. (2006) AceView: a comprehensive cDNA-supported gene and Pikuleva IA and Waterman MR (2013) Cytochromes p450: roles in diseases. J Biol Chem 288: transcripts annotation. Genome Biol 1:S12.1-14. 17091–17098. Tian D and Zhang ZQ (2011) 2 Novel deletions of the sterol 27-hydroxylase gene in a Chinese Qiao J, Han B, Liu BL, Liu W, Wu JJ, Pan CM, Jiang H, Gu T, Jiang BR, Zhu H, et al. (2011) A Family with Cerebrotendinous Xanthomatosis. BMC Neurol 11:130. unique exonic splicing mutation in the CYP17A1 gene as the cause for steroid 17alpha- Toscano C, Klein K, Blievernicht J, Schaeffeler E, Saussele T, Raimundo S, Eichelbaum M, hydroxylase deficiency. Eur J Endocrinol 164:627–633. Schwab M, and Zanger UM (2006) Impaired expression of CYP2D6 in intermediate metabo- Qu G, Dong X, Piazza CL, Chalamcharla VR, Lutz S, Curcio MJ, and Belfort M (2014) RNA- lizers carrying the *41 allele caused by the intronic SNP 2988G.A: evidence for modulation of RNA interactions and pre-mRNA mislocalization as drivers of group II intron loss from nuclear splicing events. Pharmacogenet Genomics 16:755–766. genomes. Proc Natl Acad Sci USA 111:6612–6617. Tournel G, Cauffiez C, Billaut-Laden I, Allorge D, Chevalier D, Bonnifet F, Mensier E, Lafitte JJ, Raj A and van Oudenaarden A (2008) Nature, nurture, or chance: stochastic gene expression and its Lhermitte M, Broly F, et al. (2007) Molecular analysis of the CYP2F1 gene: identification of a consequences. Cell 135:216–226. frequent non-functional allelic variant. Mutat Res 617:79–89. Rappaport N, Twik M, Nativ N, Stelzer G, Bahir I, Stein TI, Safran M, Lancet D. (2014) Tralau T and Luch A (2013) The evolution of our understanding of endo-xenobiotic crosstalk and MalaCards: a comprehensive automatically mined database of human diseases. Curr Protoc cytochrome P450 regulation and the therapeutic implications. Expert Opin Drug Metab Toxicol Bioinformatics 47:1.24.1–1.24.19 DOI: 10.1002/0471250953.bi0124s47 (published online). 9:1541–1554. Rat E, Billaut-Laden I, Allorge D, Lo-Guidice JM, Tellier M, Cauffiez C, Jonckheere N, van Trump DL, Muindi J, Fakih M, Yu WD, and Johnson CS (2006) Vitamin D compounds: clinical Seuningen I, Lhermitte M, Romano A, et al. (2006) Evidence for a functional genetic development as cancer therapy and prevention agents. Anticancer Res 26 (4A):2551–2556. Alternative P450 Splicing Redirects Human Drug Metabolism 389

Turman CM, Hatley JM, Ryder DJ, Ravindranath V, and Strobel HW (2006) Alternative splicing and differential expression of the IIB mRNAs in human liver. Biochemistry 28: within the human cytochrome P450 superfamily with an emphasis on the brain: The convolution 7340–7348. continues. Expert Opin Drug Metab Toxicol 2:399–418. Zanger UM and Schwab M (2013) Cytochrome P450 enzymes in drug metabolism: regulation of Vanselow J, Zsolnai A, Fésüs L, Fürbass R, and Schwerin M (1999) Placenta-specific transcripts of gene expression, enzyme activities, and impact of genetic variation. Pharmacol Ther 138: the aromatase encoding gene include different untranslated first exons in sheep and cattle. Eur J 103–141. Biochem 265:318–324. Zaphiropoulos PG (1997) Exon skipping and circular RNA formation in transcripts of the human Verrips A, Steenbergen-Spanjers GC, Luyten JA, Wevers RA, Wokke JH, Gabreëls FJ, Wolthers cytochrome P-450 2C18 gene in epidermis and of the rat androgen binding protein gene in testis. BG, and van den Heuvel LP (1997) Exon skipping in the sterol 27-hydroxylase gene leads to Mol Cell Biol 17:2985–2993. cerebrotendinous xanthomatosis. Hum Genet 100:284–286. Zaphiropoulos PG (1999) RNA molecules containing exons originating from different members of Walsh AA, Szklarz GD, and Scott EE (2013) Human cytochrome P450 1A1 structure and utility in the cytochrome P450 2C gene subfamily (CYP2C) in human epidermis and liver. Nucleic Acids understanding drug and xenobiotic metabolism. J Biol Chem 288:12932–12943. Res 27:2585–2590. Wang A, Stout CD, Zhang Q, and Johnson EF (2015) Contributions of ionic interactions and Zhang C, Dowd DR, Staal A, Gu C, Lian JB, van Wijnen AJ, Stein GS, and MacDonald PN (2003) protein dynamics to cytochrome P450 2D6 (CYP2D6) substrate and inhibitor binding. J Biol Nuclear coactivator-62 kDa/Ski-interacting protein is a nuclear matrix-associated coactivator that – Chem 290:5092 5104. may couple vitamin D receptor-mediated transcription and RNA splicing. J Biol Chem 278: Wang D, Poi MJ, Sun X, Gaedigk A, Leeder JS, and Sadee W (2014) Common CYP2D6 poly- 35325–35336. morphisms affecting alternative splicing and transcription: long-range haplotypes with two Zhang C, Wang L, Liao Q, Zhang L, Xu L, Chen C, Ye H, Xu X, Ye M, and Duan S (2013) – regulatory variants modulate CYP2D6 activity. Hum Mol Genet 23:268 278. Genetic associations with hypertension: meta-analyses of six candidate genetic variants. Genet Wang LH, Tazawa R, Lang AQ, and Wu KK (1994) Alternate splicing of human thromboxane Test Mol Biomarkers 17:736–742. – synthase mRNA. Arch Biochem Biophys 315:273 278. Zhao Y, Goto K, Saitoh M, Yanase T, Nomura M, Okabe T, Takayanagi R, and Nawata H (2002) Wang W, Fu JF, Gong FQ, Zhu WH, and Shen Z (2011) Rare hypertension as a result of 17alpha- – Activation function-1 domain of androgen receptor contributes to the interaction between sub- hydroxylase deficiency. J Pediatr Endocrinol Metab 24:333 337. nuclear splicing factor compartment and nuclear receptor compartment. Identification of the Wang Z and Burge CB (2008) Splicing regulation: from a parts list of regulatory elements to an – p102 U5 small nuclear ribonucleoprotein particle-binding protein as a coactivator for the re- integrated splicing code. RNA 14:802 813. ceptor. J Biol Chem 277:30031–30039. Warner SC, Finta C, and Zaphiropoulos PG (2001) Intergenic transcripts containing a novel human Zhao B and Waterman MR (2011) Moonlighting cytochrome P450 . IUBMB Life cytochrome P450 2C exon 1 spliced to sequences from the CYP2C9 gene. Mol Biol Evol 18: – – 63:473 477.

1841 1848. Downloaded from Zhou C, Zhou D, Esteban J, Murai J, Siiteri PK, Wilczynski S, and Chen S (1996) Aromatase gene Wiemann S, Kolb-Kokocinski A, and Poustka A (2005) Alternative pre-mRNA processing regu- expression and its exon I usage in human breast tumors. Detection of aromatase messenger RNA lates cell-type specific expression of the IL4l1 and NUP62 genes. BMC Biol 3:16. by reverse transcription-polymerase chain reaction. J Steroid Biochem Mol Biol 59:163–171. Wu AH (2011) Drug metabolizing enzyme activities versus genetic variances for drug of clinical Zhou R, Chun RF, Lisse TS, Garcia AJ, Xu J, Adams JS, and Hewison M (2015) Vitamin D and pharmacogenomic relevance. Clin Proteomics 8:12–21. alternative splicing of RNA. J Steroid Biochem Mol Biol (148):310–317. Wu S, Ren S, Nguyen L, Adams JS, and Hewison M (2007) Splice variants of the CYP27b1 gene Zhou SF, Liu JP, and Chowbay B (2009) Polymorphism of human cytochrome P450 enzymes and and the regulation of 1,25-dihydroxyvitamin D3 production. Endocrinology 148:3410–3418. its clinical impact. Drug Metab Rev 41:89–295. Wu Z, Puigserver P, Andersson U, Zhang C, Adelmant G, Mootha V, Troy A, Cinti S, Lowell B, Zhou SF (2009) Polymorphism of human cytochrome P450 2D6 and its clinical significance: Part I. Scarpulla RC, et al. (1999) Mechanisms controlling mitochondrial biogenesis and respiration Clin Pharmacokinet 48:689 –723. through the thermogenic coactivator PGC-1. Cell 98:115–124. dmd.aspetjournals.org Wu ZL, Bartleson CJ, Ham AJ, and Guengerich FP (2006) Heterologous expression, purification, Zimmerly S and Semper C (2015) Evolution of group II introns. Mob DNA 6:7. and properties of human cytochrome P450 27C1. Arch Biochem Biophys 445:138–146. Yamaguchi H, Nakazato M, Miyazato M, Toshimori H, Oki S, Shimizu K, Suiko M, Kangawa K, and Matsukura S (1998) Identification of a novel splicing mutation and 1-bp deletion in the Address correspondence to: Dr. Andrew J. Annalora, Department of Environ- 17alpha-hydroxylase gene of Japanese patients with 17alpha-hydroxylase deficiency. Hum Genet mental and Molecular Toxicology, Oregon State University, 1007 Agriculture and 102:635–639. Life Sciences Building, Corvallis, OR 97331. E-mail: Andrew.Annalora@oregon- Yamano S, Nhamburo PT, Aoyama T, Meyer UA, Inaba T, Kalow W, Gelboin HV, McBride OW, and Gonzalez FJ (1989) cDNA cloning and sequence and cDNA-directed expression of human state.edu P450 IIB1: identification of a normal and two variant cDNAs derived from the CYP2B locus on at ASPET Journals on October 3, 2021 SUPPLEMENTAL MATERIALS

TITLE: Alternative Splicing in the Cytochrome P450 Superfamily Expands Protein Diversity to

Augment Gene Function and Redirect Human Drug Metabolism.

Authors: Andrew J. Annalora1, Craig B. Marcus1, Patrick L. Iversen1

Journal: Drug Metabolism and Disposition

SUPPLEMENTAL DISCUSSION

The Environmental Genome Project was launched by the NIEHS to characterize variations in important genes and relate the differences to human susceptibility to chemical and physical agents in the environment (Guengerich, 1998). The project sought to investigate the significance of polymorphisms that encompass: (i) altered codon producing amino acid substitutions, (ii) premature stop codon introduction, (iii) diminished RNA transcription, (iv) splice variants, and (v) altered mRNA stability. Because of their central role in phase I drug metabolism, the characterization of SNP-sensitive, CYP variant forms, remains an important aspect of the environmental genome project. While this review highlights some of the mechanisms by which genetic polymorphisms alter CYP gene splicing, we also wanted to focus the reader on some of the intrinsic cellular splicing mechanisms that promote alternative gene assembly, and alternative exon usage, under normal conditions, enabling individualized approaches to healthcare.

OVERVIEW OF RNA PROCESSING AND ALTERNATIVE SPLICING IN HUMANS

Greater than 90% of mammalian multi-exon genes are alternately spliced (Croft et al.,

2000), and alternate splicing is linked to multiple human diseases (Cooper et al., 2009). Alternate splicing or alternate exon inclusion during pre-mRNA maturation is a process in which a single gene can encode multiple proteins. For genes with more than one exon, each intron contains conserved elements with a 5’-\GU-nn(n)xnnYUNAYYYYYYYYYYYYYYYAG\-3’ sequence. These conserved sequences are recognized by a variety of small nucleolar ribonuclear proteins

(snRNP). The U1snRNP binds AG-GU sequences at the splice donor site (U12snRNP binds AU-

AC donors) and recruits U2AF35 at the 3’-end of the intron and U2AF65 to a branch site A

(underlined), which binds to the polypryimidine tract forming the spliceosome A complex. The 2’-

OH of adenine attacks the 5’-terminus of the intron and U5snRNP positions exons for a second nucleophilic attack at the 3’-OH of the exon, joining the exon donor and exon acceptor and eliminating the intron. The cis-elements at the 5’-donor are AG/RURAGU, the 3’-acceptor are

YAG/RNNN, the branch point YNYURAC, and the polypyrimidine tract. The sequence context of the exon junctional complex (EJC) may weakly or strongly define an exon and mutations in the

5’-donor or 3’-acceptor region tend to diminish but not necessarily extinguish spliceosome recognition (Lewandowska, 2013). Additional regulation of splicing involves cis-elements referred to as intronic splicing-silencers (ISS), intronic splicing-enhancers (ISE), exonic splicing-silencers

(ESS), and exonic splicing-enhancers (ESE) and trans-acting proteins which are generally members of the serine-rich (SR) proteins (Falanga et al., 2014). A simplified, schematic diagram of the alternative splicing process is shown in Figure 1. It highlights how tissue-specific, trans- acting regulatory factors, such as the U2 and U10 snRNP, work in conjunction with cis-regulatory elements, to promote exon inclusion or exclusion (Supplemental Figure 1).

The pattern of pre-mRNA splicing can be altered by both endogenous and exogenous compounds, epigenetic modifications and with splice-switching oligonucleotides. This process is also influenced by the structure of a gene’s promoter, and the organization of various hormone- response elements that guide the recruitment of requisite transcription factors and co-regulatory factors (Auboeuf et al., 2002). For example, glucocorticoids can modulate alternative exon inclusion and are synthesized and degraded by cytochrome P450 genes that are sensitive to this process (Park et al., 2009). While the mechanism of this endocrine disruption remain poorly defined, the splice altering action of glucocorticoids appears to involve the glucocorticoid (GR) nuclear receptor (Lai and McCobb, 2002). Interestingly, the exon skipping induced by glucocorticoids is now being exploited for the treatment of Ataxia Telangiectasia (ATM) where exon 3 is joined to exon 52 of the ATM pre-mRNA (Menotta et al., 2012). The alternative splicing and expression of CYP genes that alter the balance of endogenous glucocorticoids and their interaction with the GR to alter pre-mRNA splicing, deserve greater attention. Several antisense oligonucleotides are being evaluated as therapeutic agents to treat genetic disease by altering splice site recognition in pre-mRNA (Disterer et al., 2014; see Supplemental Table 1).

Emerging epigenetic regulators of alternate splicing include DNA methylation (e.g. 5- methylcytosine formation), Argonaut (AGO1 and AGO2), and lncRNA (Zhou et al., 2013; Li et al.,

2014). Nucleosomes are preferentially positioned over exons and are enriched in specific histone modifications such as H3K35me3 and H3K79me1 (Hon et al., 2009). Elevated histone acetylation leads to increased exon skipping (Schor et al., 2010). The rate of RNA polymerase II (RNAPII) also influences splicing efficiency and exon inclusion. In turn, the structure of chromatin influences the RNAPII transcription rate and the co-transcriptional nature of splicing (Brown et al.,

2012). Chromatin organization affects alternative splicing which may involve exon-nucleosome occupancy. If the 5’-splice site pairing with U1 snRNA is strengthened then exon inclusion is observed and leads to increased nucleosome occupancy (Karam et al., 2013). Current investigations continue to reveal a more refined understanding of how epigenetic control of splicing is regulated with emphasis on age, specific tissue types, and the role of cell transformation. Alternately spliced transcripts that result in mRNA that are no longer in-frame are likely to be degraded by nonsense-mediated decay (NMD) processes. RNA degradation is carried out by over 30 families of ribonucleases, and humans encode more than 60 different isoforms.

Aberrantly processed, defective RNA molecules can damage cells, thus creating a need for diverse surveillance and degradative pathways (Stoecklin and Muhlemann, 2013). Cellular mRNA decay utilizes multiple strategies, and NMD will act on mRNA with an ORF-interrupting premature termination codon (PTC), upstream ORFs (uORFs), introns in the 3’-UTR, long 3’- UTRs, and alternative polyadenylation or poly(A) mutations (Dickson and Wilusz, 2011).

Common NMD substrates have translation termination that is distant from the poly(A) tail, or possess an exon junction complex (EJC) between the stop codon and the poly(A) tail. PTCs located >50-55 nucleotides upstream of a 3’ EJC trigger NMD (Schweingruber et al., 2013).

Staufin-1 (Stau1)-mediated mRNA decay (SMD) can also recruit upframeshift protein 1 (UPF1) to the EJC, similar to NMD, but involves transcript binding, not splicing (Karam et al., 2013). The

ATPase and RNA helicase activity of UPF1 and related (Upf2 and Upf3’s) target mRNA and nascent peptide for degradation and may facilitate ribosome subunit dissociation and recycling events (Celik et al., 2014). STAU1 binding sites can be formed by Alu elements in 3-UTRs and lncRNA, and binding to these sites induces SMD (Gong and Maquat, 2011). Fully modified morpholino and 2’-O-methyl antisense oligomers that are not substrates for RNase H or

RISC activity can lead to RNA degradation through NMD, as well (Ward et al., 2014).

During protein elongation the pairing of tRNA anti-codons and mRNA codons occurs at the ribosomal A site accompanied by stringent proofreading steps that ensure highly accurate protein synthesis. Suppression of termination occurs at a rate of 0.001 – 0.1 percent at normal stop codons and 0.01 – 1 percent at premature termination codons (PTC). Suppression of stop codons is mediated by aminoacyl-tRNA mispairing with a stop codon and amino acids tryptophan, tyrosine and lysine have been observed to be present at the PTC site indicating near-cognate mispairing can occur at any of the three codon positions (Fearon et al., 1994). Codons in mRNA include UGA as a leaky stop, UAA as a strong stop and UAG as an intermediate (Weiner and

Weber, 1973). The 16S rRNA (eukaryotic 18S) bases A1492 and A1493 (eukaryotic A1754 and

A1755) flip out of the helix to interact with the minor groove of the codon-anticodon helix in site A of the ribosome, and G530 flips from syn- to an anti- conformation. These interactions confirm fidelity and cause the 30S subunit to shift from open to closed conformation and GTP hydrolysis by EF-Tu (Keeling et al., 2012). Aminoglycosides bind to 16S rRNA near A1492 and

A1493 displacing bases to the minor groove of helix 44 resulting in translation read-through and suppression of premature codon termination (Lai et al., 2004). Therapeutics such as Ataluren

(Peltz et al., 2013) have been designed to suppress PTCs in genetic diseases including cystic fibrosis, Duchenne muscular dystrophy, ataxia telangiectasia, recessive dystrophic epidermolysis bulosa, and Hurler syndrome, but still face limitations linked to insufficient efficacy, incorporation of nonfunctional amino acids at PTCs, NMD, and both toxicity and immune responses linked to neoantigens resulting from therapy (Keeling et al., 2012). Ultimately, the role P450 enzymes play in the degradation of aminoglycosides and related compounds imply a potentially central role for

CYP metabolism in the regulation of protein translation, particularly for SNP-based or alternatively-spliced transcripts subject to NMD.

Based on the global nature of the splicing process, it is not surprising that nearly all 57 human CYP genes are subject to alternative splicing events, via one of seven traditional alternative splicing mechanisms operating in metazoan genes (Blencowe, 2006; Roy et al., 2013).

However, the ability of CYPs to metabolize both endogenous and exogenous modulators of gene splicing and protein translation (e.g. glucocorticoids, cyclohexamide, aminoglycocides, etc.) appears to be under-appreciated in the literature (Busi and Cresteil, 2005). This review highlights how our modern understanding of alternative splicing shatters the classical one gene-one transcript (or polypeptide) paradigm (Beadle and Tatum; 1941; Chow et al., 1977; Davis, 2007), and suggests that each gene encodes multiple coding and noncoding transcripts to ensure the expression of the most adaptive phenotype. This observation helps explain both the apparent paucity of coding information in the human genome and the potential role for the increasingly visible assemblage of non-coding RNA “dark matter” that was associated with “junk DNA” only a decade ago. Below we review some of the best examples of alternative P450 gene splicing in humans to highlight the varied cellular mechanisms regulating their expression. We will also focus in on how improvements in structural and computational biology have shifted our appreciation for

CYP splice variants and their biological significance with time.

SUPPLEMENTAL REFERENCES

Auboeuf D, Dowhan DH, Kang YK, Larkin K, Lee JW, Berget SM, and O’Malley BW (2002) Coordinate regulation of transcription and splicing by steroid receptor coregulators. Science 298(5592): 416-419.

Beadle GW, Tatum EL (1941) Genetic Control of Biochemical Reactions in Neurospora. Proc Natl Acad Sci U S A 27(11): 499-506.

Blencowe BJ (2006) Alternative splicing: new insights from global analyses. Cell 126(1): 37-47. Review.

Brown SJ, Stoilov P, and Xing Y (2012) Chromatin and epigenetic regulation of pre-mRNA processing. Hum Mol Genet 21(R1): R90-R96.

Busi F, Cresteil T (2005) CYP3A5 mRNA degradation by nonsense-mediated mRNA decay. Mol Pharmacol 68(3): 808-15.

Celik A, Kervestin S, Jacobson A (2015) NMD: At the crossroads between translation termination and ribosome recycling. Biochimie 114: 2-9. Review. Chow LT, Gelinas RE, Broker TR, Roberts RJ (1977) An amazing sequence arrangement at the 5' ends of adenovirus 2 messenger RNA. Cell 12(1): 1-8. Cooper TA, Wan L, Dreyfuss G (2009) RNA and disease. Cell 136(4): 777-93. Review.

Croft L, Schandorff S, Clark F, Burragge K, Arctander P, and Mattick JS. (2000) ISIS, the intron information system, reveals the high frequency of alternative splicing in the human genome. Nat. Genet 24: 340-341.

Davis RH (2007) Beadle's progeny: innocence rewarded, innocence lost. J Biosci 32(2): 197-205.

Dickson AM and Wilusz J (2011) Strategies for viral RNA stability: live long and prosper. Trends Genet 27: 286-293.

Disterer P, Kryczka A, Liu Y, Badi Y, Wong JJ, Owen JS, and Khoo B (2014) Development of Therapeutic Splice-Switching Oligonucleotides. Human Gene Therapy 25: 587-598.

Falanga A, Stojanovic O, Kiffer-Moreira T, Pinto S, Millan JL, Vlajovicek K, and Baralle M (2014) Exonic splicing signals impose constraints upon the evolution of enzymatic activity. Nucl Acid Res 42: 5790-5798.

Fearon K, McClendon V, Bonetti B and Bedwell DM (1994) Premature translation termination mutations are effectively suppressed in a highly conserved region of yeast Ste6P, a member of the ATP-binding cassette (ABC) transporter family. J Biol Chem 269: 17802-17808.

Gong C and Maquat LE (2011) lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3’-UTRs via Alu elements. Nature 470: 284-290.

Guengerich FP (1998) The Environmental Genome Project: Functional Analysis of Polymorphisms. Environment Health Perspect 106(7): 365-368. Hon G, Wang W and Ren B (2009) Discovery and annotation of functional chromatin signatures in the human genome. PLOS Comput. Biol 5: e1000566.

Karam R, Wengrod J, Gardner LB, and Wilkinson MF (2013) Regulation of nonsense-mediated mRNA decay: Implications for physiology and disease. Biochem et Biophys Acta 1829:624-633.

Keeling KM, Wang D, Conard SE and Bedwell DM (2012) Suppression of premature termination codons as a therapeutic approach. Crit Rev Biochem Mol Biol 47(5): 444-463.

Lai GJ and McCobb DP (2002) Opposing actions of adrenal androgens and glucocorticoids on alternative splicing of Slo potassium channels in bovine chromaffin cells. Proc Natl Acad Sci 99(11): 7722-7727.

Lai CH, Chun HH, Nahas SA, Mitui M, Gamo KM, Du L, and Gatti RA (2004) Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons. Proc Natl Acad Sci 101(44): 15676-15681.

Lewandowska MA (2013) The missing puzzle piece: splicing mutations. Int J Clin Exp Pathol 6(12): 2675-2682.

Li X, Wu Z, Fu X, Han W (2014) lncRNAs: insights into their function and mechanics in underlying disorders. Mutat Res Rev Mutat Res 762: 1-21.

Menotta M, Biagiotti S, Chessa BL and Magnani M (2012) Dexamethasone partially rescues Ataxia Telangiectasia-mutated (ATM) deficiency in Ataxia Telangiectasia by promoting a shortened protein variant retaining kinase activity. J Biol Chem 287: 41352-41363.

Park E, Lee ML, Baik SM, Cho EB, Son GH, Seong JY, Lee KH, and Kim K (2009) NOVA-1 mediates glucocorticoid-induced inhibition of pre-mRNA splicing of gonadotropin-releasing hormone transcripts. J Biol Chem 284: 12792-12800.

Peltz SW, Morsy M, Welch EM, and Jacobson A (2013) Ataluren as an agent for therapeutic nonsense suppression. Ann Rev Med 64: 407-425.

Roy B, Haupt L, and Griffiths LR (2013) Review: Alternate splicing (AS) of genes as an approach for generating protein complexity. Curr Genomics 14: 182-194. Schor IE, Allo M, and Kornblihtt AR (2010) Intragenic chromatin modifications: a new layer in alternative splicing regulation. Epigenetics 5: 174-179. Schweingruber C, Rufener S, Zund D, Yamashita A and Muhlemann O (2013) Nonsense- mediated mRNA decay- mechanisms of substrate mRNA recognition and degradation in mammalian cells. Biochem et Biophys Acta 1829: 612-623.

Stoecklin G and Muhlemann O (2013) RNA Decay mechanisms: Specificity through diversity. Biochem et Biophys Acta 1829:487-490.

Ward AJ, Norrgom M, Chun S, Bennett CF, and Rigo F (2014) Nonsense-mediated decay as a terminating mechanism for antisense oligonucleotides. Nucl Acids Res 42: 5871-5879.

Weiner AM and Weber K (1973) A single UGA codon functions as a natural termination signal in the coliphage q beta coat protein cistron. J Mol Biol 80: 837-855.

Zhou HL, Luo G, Wise JA, and Lou H (2013) Regulation of alternate splicing by local histone modifications: potential roles for RNA-guided mechanisms. Nucleic Acids Res 42(2): 701-713.

SUPPLEMENTAL TABLES

Supplemental Table 1. CYP Transcript Variant Subcellular Localization

Family Cytoplasm ER Mitochondria Membrane Nucleus Secreted 1A1-251# 1A1- 1A1- CYP1 1B1-127*† ∆Ex6 ∆Ex2* 2A6-494* 2A7-494, 2A7-101*† 443 2C8-103*† 2A13-494‡ 2E1-335* 2C8-490*, 2F1-125* 404*,307* 2J2-312 2C8-388 2C9-490*, CYP2 2R1-386*, 2E1-252† 282,101 228,158*† 2E1-493 2S1-373*, 2J2- 229 502,402, 2W1-198* 355 2R1-501 2U1-596* 3A4-423 3A4-473* 3A5-228† 3A5-389* 3A4-516*, 3A7-423 3A7-473* 503, 502 CYP3 3A43-393, 3A43- 3A5-502*, 280,250, 504,503, 492*,322* 248,229† 420 4B1-512*, 4F2- 4A22-519, CYP4 4A11-220† 4X1-509 511,325 190*† 455, 357 4B1-449, 4F8-194† 4F2-520*, 194† 4F22-531* 420,162 4F2-371 4V2-525 4F8-155 4F3-371*, 4F11-524* 230 4F12-524, 4F8-347, 229,172, 305,245, 102† 195,183† 4V2-178† 4F11- 308*† 4F12-314, 273,231, 138† 4V2-197, 171† 4X1-293 CYP7 7A1-504*‡ CYP8 8A1-361 11A1-512, 469, 460, 11B1- CYP11 439, 433, 11B1-154 11B1-113† 153* 414†, 250

19A1-344 CYP19 19A1-129* 19A1-271

CYP20 20A1-234 20A1-208

21A2-495, 21A2-422, CYP21 465*, 373, 401, 347 239 CYP24 24A1-372 24A1-448* 26A1-497* CYP26 26A1-151† 26C1-302† 27A1- CYP27 27C1-372 27A1-115† 395* 39A1- CYP39 449* CYP46 46A1-337 46A1-500* CYP51 51A1-449* 51A1-509 # CYP gene name (in bold) followed by variant transcript length in base pairs. * Associated with tumor or transformed cells. † Unknown Protein Coding Status; Conflict among Ensembl or Aceview Databases; Transcript has “Protein Quality” score below “Very Good”. Potential NMD candidate. ‡ Represents the only known transcript.

Supplemental Table 2. Therapeutic Applications of Splice-Switching Oligonucleotides

Gene Disease Target Details/Expression Change

Eteplirsen; Improved 6 Minute Duchenne Muscular Dystrophin ∆ Exon 51 Walk Test (MWT); Reduced Dystrophy Creatine phosphokinase

Spinal muscular Intrathecal 9 mg dose SMN1 ISS - intron 7 atrophy Includes exon 7

LMNA Hutchinson-progeria Exon 10 - SA 25-mer PMO full length LMNA

STAT3 Cancer Exon 23 - ESE Switch STAT3a to STAT3b

Membrane bound to soluble FLT1 Neovascularization Exon 13 - SA FLT1

Fukuyama Cong. Rescue normal fukutin FKTN ISE +3’ - SA Musc. Dyst. expression

USH1C Usher syndrome Cryptic 5’ SD Rescue hair cells in cochlea

Hypertrophic MYBPC3 ESE - exon 5+6 Increase variant 4 mRNA cardiomyopathy Corneal graft Membrane bound to soluble KDR Exon 13 5’ SD rejection KDR

Familial APOB Int 27 - SA Skip exon 27 hypercholesterolemia

SUPPLEMENTAL FIGURES

Supplemental Figure 1. Schematic Diagram of the Alternative Gene Splicing Process – The function of a multi-exon gene can be manipulated post-transcriptionally by altering the exon composition of the processed mRNA transcript. All introns contain a conserved 5’-\GU- nn(n)xnnYUNAYYYYYYYYYYYYYYYAG\-3’ sequence, which like exons, also contain cis-acting regulatory elements that guide the splicing process. Trans-regulatory factors, including serum response factors (SRF), heterogeneous nuclear ribonucleoproteins (hnRNP) and small nuclear ribonucleoproteins (snRNP) interact with cis-elements in the gene that either enhance, or silence, the spliceosome’s ability to recognize an exon, and properly assemble near the splice junction.

Well-defined exons, marked by SRF recognition, contain intronic enhancer elements that promote normal recruitment of the U2 snRNP to the A branch site. Poorly-defined exons may be partially masked from the spliceosome via interactions with trans-acting elements in the exon (e.g. hnRNPs). Proper splicing also relies on coordinated interactions among multiple trans-acting silencer at (+) and (-) cis-elements in the intron. In these complex scenarios, recruitment of an intronic silencer can occlude branch site recognition by the U2 snRNP. Tissue-specific expression of a second snRNP (e.g. U10 snRNP), which can still recognize the A branch-site in the presence of intronic silencers, would be required to promote normal splicing of the reference gene transcript. In the absence of U10snRNP, or another compensatory trans-acting element, the third exon would routinely be skipped allowing the splice variant transcript to accumulate.

Supplemental Figure 2. Alternative Splicing and Function of the CYP1B1 Gene – CYP1B1 is unique among CYP1 family P450 genes in that its polypeptide sequence is translated from only

2 coding exons (exons 2 and 3); related CYP1A genes typically have 7 exons. SNPs found in the CYP1B1 gene of Glaucoma patients promote can produce truncated (or deletion) variants, including verified forms of 93 or 354 amino acid residues (Tanwar et al., 2009). The potential for cryptic exon 1 usage, and documented insertions (and deletions) in exon 2 (Sheikh et al., 2014) provide this primitive gene some structural plasticity near its N-terminus, which is extended by

~10 residues from related CYP1A genes. The extended C-terminus of CYP1B1, which is ~20 amino acids longer than CYP1A1, remains poorly characterized, with an undetermined functional role. While the evolution of such variability in CYP gene structure remains a mystery reaching back over 500 million years, a mechanism for prioritizing protein translation prioritization may be feasible, as the nuclear proteins associated with splice junction-site recognition at intron/exon borders are now known to augment the rate of protein translation in the cytoplasm (Lejeune and

Maquat, 2005; McGlincy and Smith, 2008).

Supplemental Figure 3. Alternative-Splicing of the Human CYP19A1: Tissue-specific Exon

1 and 5 Inclusion - Exon 1 of the CYP19A1 gene is highly complex, and is organized into 5 cryptic exons (I.1, I.4, I.2, I.3 & PII) subject to tissue-specific, alternate splicing in mammals (Zhou et al., 1996). CYP19A1 variants with reduced aromatase activity have been detected in adipose tissue, brain, testis, adrenal glands and bone (Lin et al., 2007). Structural analysis of CYP19A1

(PDB: 3S79) indicates alternative exon 1 usage could alter membrane binding, substrate access and subcellular distribution by altering the structure of the N-terminus encoded by Exon 2 (green).

More recently, it was determined that exon 5 in the CYP19A1 gene is poorly defined and subject to both splicing mutations and physiological alternative splicing events (Pepe et al., 2007). The skipping of exon 5 (yellow ribbon), which encodes complete α helices D and E in CYP19A1, eliminates prototypical P450 aromatase activity in both steroidogenic and non-steroidogenic tissues (Lin et al., 2007; Pepe et al., 2007). The modular organization of these discrete structural elements into exon 5, suggests a domain swapping mechanism underlying the aromatization process, based on tissue-specific exon 5 usage.

Supplemental Figure 4. Alternative Splicing of Human Drug Metabolizing P450s: CYP2C9 and CYP2D6. A.) Several CYP2C9 splice variants (CYP2C9sv) have been identified (Ohgiya et al., 1992), including a liver-specific form that skips exon 2 that is spectrally-active, but does not metabolize prototype 2C9 substrates (Ariyoshi et al., 2007). Structural analysis of CYP2C9

(PDB: 4NZ2) indicates removal of exon 2 (in yellow ribbon), and a partial insertion of intron 1, would reshape the substrate access channel and membrane binding surface by altering the N- terminus, helices A’-A, and β sheets 1 and 2. These changes could also reshape the distal pocket of the active site and reposition the proximal meander region, altering redox partner binding interactions and catalytic function. The import of liver-specific “plug-and-play” usage of exon 2 in

CYP2C9 remains enigmatic, but relatively ubiquitous, making it a possible marker of disease or metabolic phenotype. The CYP2C9 gene, located within a cluster of CYPs on chromosome

10q24 is also subject to non-random, trans-splicing events with neighboring CYPs 2C8, 2C18 and

2C19 in liver and skin (Warner et al., 2001); multiple chimeric variants, many containing exon 1 from CYP2C18 spliced onto exons from CYP2C9 have been detected, and could play a similar role in altering substrate recognition or catalytic function. B.) The human CYP2D gene family is also complex, comprised of CYP2D6 and 4 pseudogenes (CYP2D7P1 & 2 and CYP2D8P1 & 2).

Alternative splicing has been detected in human liver and breast tissue (Huang et al., 1997), including a highly-expressed variant which completely skips exon 6. Analysis of the CYP2D6 crystal structure (PDB: 4WNU) reveals that exon 6 (in yellow ribbon; H-I loop – Helix J) encodes the complete I helix of CYP2D6, and removal of this large central helix may have profound metamorphic effects on the P450 fold. Alternate exon usage in CYP2D6 is also tissue-selective and variants skipping exon 3 (in orange; B’-C loop – D-E loop) and portions of exon 4 (in magenta;

D-E loop – G’ helix) have been documented that alter both enzyme function and localization

(Sangar et al., 2010). Intronic SNPs in CYP2D6*41 individuals are also known to increase the expression levels of 2D6 variants lacking exon 6 (Toscano et al., 2006). Multiple splicing mechanisms appear to underlie the complex genotype-phenotype relationship of the CYP2D6 gene, and various tissue- and transformation-sensitive variants appear to be involved.

Supplemental Figure 5. Trans-Splicing of the Human CYP3A Gene Family: Cryptic

Alternative Exon 1 Usage. There are four CYP3A genes in humans (3A4, 3A5, 3A7 & 3A43), each consisting of 13 coding exons sharing 71-88% amino acid identity that cluster on chromosome 7q21-22.1, where the CYP3A43 gene is in a head-to-head orientation with the other three genes (Finta and Zaphiropoulos, 2000). Several chimeric forms of CYP3A have been described where exon 1 of CYP3A43 is joined at canonical splice sites of CYP3A4 and CYP3A5 genes, implying a common trans-splicing mechanism. A) Our structural analysis of CYP3A4

(PDB: 4K9T) highlights the complex structural segmentation of the CYP3A4 gene into 13 exonic structures, which may facilitate its rapid transcription and translation upon xenobiotic induction.

This modular assembly may also allow the cell to sample increased CYP3A4 conformational space when attempting to adapt the catalytic site to novel environmental challenges. B.) Primary sequence alignments of exon 1 demonstrate that each human CYP3A gene encodes a distinct protein sequence, allowing trans-splicing events to alter determinants of membrane binding, substrate recognition, and subcellular targeting. C.) A CYP3A4 trans-splicing variant containing exon 1 of CYP3A43, followed by exons 4-13 of CYP3A4, has been described that retains detectable 6-β-hydroxylase activity against testosterone despite losing key structural features encoded by exons 2 and 3. D.) An additional CYP3A4 trans-splicing variant containing exon 1 of CYP3A43, followed by exons 7-13 of CYP3A4, has also been studied; this severely-truncated variant initiates coding near helix E and displays minimal 6-β-hydroxylase activity but is insensitive to NMD in the liver, and therefore may contribute to an adaptive CYP3A family cloud.