Alternative Splicing in the Cytochrome P450 Superfamily Expands Protein Diversity To

Home , Aldosterone synthase, CYP20A1, CYP26A1, CYP26B1, CYP26C1, CYP27C1

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Alternative Splicing in the Cytochrome P450 Superfamily Expands Protein Diversity to

Augment Gene Function and Redirect Human Drug Metabolism.

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

Department of Environmental and Molecular Toxicology, Oregon State University, 1007 Downloaded from Agriculture & Life Sciences Building, Corvallis, OR 97331; USA – AJA, CBM & PLI

dmd.aspetjournals.org

at ASPET Journals on September 30, 2021

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Running Title: Alternative CYP Splicing Redirects Human Drug Metabolism.

Corresponding author and person to whom reprint requests should be addressed:

Andrew J. Annalora, Ph.D

Department of Environmental and Molecular Toxicology

Oregon State University

1007 Agriculture & Life Sciences Building Downloaded from Corvallis, OR 97331; USA

Phone: 541-737-2706

Fax: 541-737-0497 dmd.aspetjournals.org e-mail: [email protected]

Text Pages: 29 at ASPET Journals on September 30, 2021 Number of Tables: 2

Figures: 4

References: 195

Word Count:

Abstract: 247

Introduction: 562

Discussion: 8,859

Abbreviations:

AR - androgen receptor

CYP - cytochrome P450

ESS - exonic splicing silencers

ESE - exonic splicing enhancers

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

GWAS - genome wide association studies hnRNPs - heterologous nuclear ribonuclear proteins hnRNPC - heterogeneous nuclear ribonucleoprotein C

ISS - intronic splicing silencers

ISE - intronic splicing enhancers kDa - kiloDaltons mRNA - messenger RNA Downloaded from NIH - National Institutes of Health

NMD - nonsense mediated decay

P450 - cytochrome P450 dmd.aspetjournals.org

PGx - pharmacogenomics

PPAR - peroxisome proliferator-activated receptors pre-mRNA - precursor or unprocessed messenger RNA at ASPET Journals on September 30, 2021 PTC - premature termination codon

RNA - ribonucleic acid

RNAseq - RNA sequencing

RAR – retinoic acid receptor

RXR - retinoid X receptor

SNP - single nucleotide polymorphism snRNA - small nuclear RNA snRNP - small nucleolar ribonuclear proteins

SRF – splicing regulatory factor

VDR - vitamin D receptor

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

ABSTRACT

The human genome encodes 57 cytochrome P450 (CYP) genes whose enzyme products metabolize hundreds of drugs, thousands of xenobiotics and unknown numbers of endogenous compounds including steroids, retinoids and eicosanoids. Indeed, CYP genes are the first line of defense against daily environmental chemical challenges in a manner that parallels the immune system. Several NIH databases, including PubMed, AceView, and Ensembl, were queried to establish a comprehensive analysis of the full human CYP transcriptome. This review describes Downloaded from a remarkable diversification of the 57 human CYP genes, which may be alternatively processed into nearly 1000 distinct mRNA transcripts to shape an individual’s CYP proteome. Important

CYP splice variants from families: 1A, 1B, 2C, 2D, 3A, 4F, 19A and 24A have now been dmd.aspetjournals.org documented, with some displaying alternative subcellular distribution or catalytic function directly linked to a disease pathology. The expansion of CYP transcript diversity involves tissue-specific splicing factors, transformation-sensitive alternate splicing, trans-splicing between gene at ASPET Journals on September 30, 2021 transcripts, single-nucleotide polymorphisms, and epigenetic regulation of alternate splicing.

Homeostatic regulation of variant CYP expression also is influenced by nuclear receptor signaling, suppression of nonsense mediated decay or premature termination codons, mitochondrial dysfunction or host infection. This review focuses on emergent aspects of the adaptive gene splicing process, which when viewed through the lens of CYP-NR gene interactions, resembles a primitive immune-like system that can rapidly monitor, respond and diversify to acclimate to fluctuations in endo-xenobiotic exposure. Insights gained from this review should aid future drug discovery and improve therapeutic management of personalized drug regimens.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

INTRODUCTION

The human genome sequence was completed in 2001 (Lander et al., 2001), helping to usher in the “post-genomic era” of personalized medicine. Over a decade later, however, the promise of pharmacogenomics (PGx) has yet to fully materialize, and the composition of the human genome remains enigmatic, as numerous questions linger concerning the complexity of its content and organization. For example, how can a genome with only 22,000 genes produce a proteome with over 200,000 distinct proteins? While transcription is a relatively well understood Downloaded from phenomenon, the mechanisms involved in regulating alternative pre-mRNA splicing, the driving force behind both transcriptome and proteome expansion, remains less appreciated. The ability

to link a single gene to its full suite of RNA transcripts and polypeptide products will improve our dmd.aspetjournals.org ability to assess the functional nature of its role in both physiological and pathological conditions

(La Cognata et al., 2014). The spectrum of alternate splicing mechanisms underlying the expansion of the proteome involves the use of seven main splicing types, as described at ASPET Journals on September 30, 2021 (Blencowe, 2006; Roy et al., 2013). The molecular basis for these mechanisms are complex, and beyond the scope of this review, however a brief description of the phenomenon and its key players is provided in the Supplemental Materials (see Supplemental Figure 1). Here we will focus primarily on the spectrum of phenotypic outcomes induced by alternative transcript splicing, to highlight the array of splice-sensitive features operating in the cytochrome P450 (CYP or P450) superfamily, a collection of 57 human genes that coordinate the metabolism of both drugs and endo-xenobiotics.

This work focuses new attention on the biological impact of alternative CYP gene splicing, an underappreciated component of phase I drug metabolism that may complicate or disrupt personalized approaches to medicine. Precision medicine, therefore, through the universal application of genetics, still faces many challenges beyond cost, ethical considerations, and the need for additional, whole genome sequences. Multiple resources have now been developed, in addition to the Human Genome Project, to address these challenges, including: the Hapmap

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Project, which enables single nucleotide polymorphism (SNP) arrays for >100,000 SNPs; the

1000 Genomes Project; the ENCODE project for non-coding RNAs; and the National Human

Genome Research Institute (NHGRI) genome-wide association studies (GWAS) catalogue.

GWAS have now identified two CYPs that represent a biomarker for disease or a drug response phenotype, including: (i) A CYP2C8 association with bisphosphonate-related osteonecrosis of the jaw in multiple myeloma (Sarasquete et al., 2008); and (ii) A CYP2C19 association with clopidogrel inter-individual variation as an antiplatelet drug (Shuldiner et al., 2009). Aside from Downloaded from showing strong associations, few pharmacogenetic biomarkers of this sort have successfully transitioned from discovery to clinical practice (Carr et al., 2014). Many GWAS studies have failed

to provide prognostic value because they are not designed to evaluate how variant gene dmd.aspetjournals.org expression is influenced by both cellular and environmental splicing factors. It is in this regard, that we feel a new appreciation for the complexity of alternative splicing is needed. For not only is the world of both coding and non-coding RNA more diverse and more complex than we could at ASPET Journals on September 30, 2021 have imagined even a decade ago, it is teeming with untold amounts of RNA “dark matter” and other relics of the RNA world, whose structure and function must be fully appreciated and classified, before a new guiding orthodoxy for “individualized medicine” can be safely conceived

(St. Laurent et al., 2014; Cowie et al., 2015).

META-ANALYSIS OF ALTERNATIVE GENE SPLICING IN THE CYTOCHROME P450

SUPERFAMILY

Alternative splicing regulated by tissue specific factors is a means of adapting to physiologic demands. Tissue-specific splice variants may arise from tissue-specific promoter elements (Wiemann et al., 2005) or tissue-specific splicing regulatory elements (Black, 2000).

Exon and intron definition requires interaction of discrete cis-elements (ESE, ESS, ISS, and ISE) with tissue-specific, trans-acting, splicing regulatory factors (SRF). In this way the same pre- mRNA transcript can be processed into tissue-specific, alternately spliced forms (Wang and

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Burge, 2008). The cytochrome P450 superfamily of genes is well studied from many perspectives

(Guengerich, 2013; Johnson and Stout, 2013; Pikuleva and Waterman, 2013). As of late 2015,

Pubmed listed over 23,790 citations regarding human P450 genes (according to Genecards.org), with much of this work being focused on tissue-specific expression or catalytic function of individual, reference forms. Alternative splicing of P450s has also been well-studied for more than two decades, however, the structural and functional diversity of CYP splice variants, and their relationship to human health and disease, remains poorly understood. A summary of known Downloaded from CYP splice variants linked to human disease is presented in Table 1. Reports from groups attempting to synthesize a global view of alternative P450 splicing, and its relevance to our

understanding of human metabolism in the context of PGx or personalized medicine, are dmd.aspetjournals.org exceedingly rare (Nelson et al., 2004; Turman et al., 2006).

Our review has led us to conclude that personalized approaches to medicine based solely on GWAS may be misleading, in that they fail to account for variability in compensatory splicing at ASPET Journals on September 30, 2021 mechanisms, which can modulate the expression of some highly-penetrant mutations. This phenomenon is exemplified by ontogenetic regulatory factors that can mask the physiological impact of genetic variations among developmentally-regulated CYP genes (Hines and McCarver,

2002). To expand upon this concept, and explore the array of alternative splicing products that serve as an improved platform for predicting disease based on the functional genome, we annotated all of the known human CYP splice variants currently listed in the NCBI’s Pubmed,

Aceview and Ensembl databases (Thierry-Mieg and Thierry-Mieg, 2006; Cunningham et al.,

2015) as of late 2015. Based on this computational meta-analysis, we found the 57 human CYP genes produce a P450 “transcriptome cloud” composed of at least 965 unique mRNA transcripts

(Table 2). This number represents all known reference (or wild-type) and variant CYP mRNAs including some with retained introns, premature termination codons (PTCs), and those subject to nonsense mediated decay (NMD); we also included several experimentally-derived transcripts described in Pubmed-listed citations (identified by searching CYP* or P450 splice variant or

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 alternative CYP* or P450 splicing), that were not represented in the Aceview or Ensembl databases.

Our results approximate that the average CYP gene encodes nearly 20 unique mRNA transcripts, capable of yielding both reference and splice variant proteins, and an ensemble of non-coding RNA molecules. Over 53% of these transcripts (515) are predicted to express viable proteins, based on the most stringent expression criteria utilized by both the Aceview and

Ensembl databases (Table 2). Subsequent analysis of the GeneCards.org website revealed that Downloaded from the 57 human CYPs are associated with over 48,384 SNPs, approximating to roughly 850 mutations per CYP gene (Stelzer et al., 2011). While most CYPs fall within this general range of

genetic variation, some, including CYP 2C19, 5A1, 19A1 and 39A1 express more than twice as dmd.aspetjournals.org many SNPs than average, suggesting the accumulation process is non-random (Table 2). The total number of diseases associated with each of the 57 human genes (1057) was also calculated using the Malacards.org website (Rappaport et al., 2014). CYP families 1 (203) and 2 (413) at ASPET Journals on September 30, 2021 account for over half of the total disease associations, while orphan P450s CYP20A1 (1) and

CYP39A1 (2) are associated with the fewest.

A side-by-side, radar plots comparison of the total number of alternative CYP splice variants and SNPs, for each of the 57 human CYP genes, is shown in Figure 1.

Pharmacologically-relevant drug targets, like CYP2C9, CYP2D6, CYP3A5, CYP11A1 and

CYP19A1, are among the most highly-spliced P450 genes, the majority of which are also subject to trans-splicing events. While differences in gene organization or cell-specific, selection pressure may play a role in this phenomenon, our observations suggest that drug-dosing guidelines derived from knowledge of a patient’s individual CYP transcriptome may be far superior to those based on genotyping alone, as SNP-based approaches may tend to overstate (or oversimplify) the biological influence of a point mutation on gene function. While computational studies indicate that 85% of all human mRNA comes from a single major transcript (Gonzales-Porta et al., 2013), the CYP superfamily of genes is highly diverse at the genomic level, making it uniquely-sensitive

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 to alternate splicing events that alter the biological output of both genetic and epigenetic signaling cascades. Nuclear receptors are now recognized for their role in mediating global gene splicing events (Auboeuf et al., 2005), and an improved framework for how this process functions to regulate drug metabolism appears to be in order, one that can potentially re-contextualize the utility and complexity of the convoluted metabolic cross-talk network that underlies the 48 NR signaling pathways operating in humans (Meyer, 2007; Tralau and Luch, 2013).

Furthermore, drug metabolizing P450s of CYP families 1-4, have traditionally been Downloaded from considered more polymorphic than CYPs that metabolize endogenous, cholesterol-based substrates, and are assumed to be more sensitive to SNP-based modulation of alternative splicing

(Lewiñska et al., 2013). Based on our analysis, it does appear that that the majority of steroid- dmd.aspetjournals.org metabolizing P450s, including CYP17A1 (91 SNPs) and CYP21A2 (173 SNPs), are among the least polymorphic P450 genes. As described above, mutations in these CYP enzymes are closely linked to congenital defects such as pseudohermaphroditism and adrenal hyperplasia at ASPET Journals on September 30, 2021 (Yamaguchi et al., 1998; Doleschall et al., 2014), and SNPs may be less well tolerated in these systems due to their central role in the production of sex hormones, progestins and corticoids. In contrast, the 35 drug metabolizing P450s that comprise CYPs family 1-4 in humans, average over

700 SNPs per isoform, and the 25,659 total polymorphisms in this group represent over 53% of the total P450 SNP population. While CYP2C9 (1422 SNPs), CYP2C18 (1230 SNPs) and

CYP2C19 (2467) are the most polymorphic drug-metabolizing P450s, they are considerably less polymorphic than genes like CYP5A1 (5274 SNPs) which metabolizes prostaglandins, and

CYP7B1 (3400 SNPs), which converts cholesterol to bile acids. CYP5A1 is also known as thromboxane synthase (TBXAS1), a functionally-distinct P450 that catalyzes molecular rearrangements of its substrates (Hecker and Ullrich, 1989). CYP5A1-based SNPs represent more than 10% of all CYP polymorphisms in humans and are recognized for altering tissue- specific splicing in blood and lung cells (Wang et al., 1994), and for promoting alternative exon 12 inclusion to promote cerebrovascular disease (Kimouli et al., 2009). CYP2E1 displayed the

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 lowest number of SNPs (33), and the highest alternative transcript-to-SNP ratio (0.73) among human CYP genes (see Figure 1). CYPs 21A2 (0.33), 27B1 (0.15) and CYP2D6 (0.11) also skew above normal CYP superfamily genes with respect to the average alternative transcript-to-SNP ratio of 0.05. Improved understanding of how key SNPs interact with the environment to alter individual CYP gene splicing outcomes, therefore, could be very helpful in improving the predictive power of personalized medicine based on genetic screening.

Therefore, while some SNPs alter gene function by modulating alternative splicing events, Downloaded from the total number in a population may be irrelevant from an evolutionary perspective, if they do not manifest in alternative phenotypes. Furthermore, it is intriguing that several steroid hormone-

metabolizing P450s, including CYP7B1 (3400 SNPs), CYP19A1 (2178 SNPs), and CYP39A1 dmd.aspetjournals.org

(2129 SNPs) display disproportionate amounts of polymorphisms compared to all other human

CYPs, reiterating the possibility that SNP expansion among P450 genes is neither random, nor predictable, based on the tissue-specific expression or substrate-selectivity of an individual CYP. at ASPET Journals on September 30, 2021 What may be more important with respect to the accumulation of SNPs may be their ability to be silenced or masked via alternative- or trans-splicing paradigms that render them invisible or inconsequential to the natural selection process. The defective nature of a CYP-related SNP may depend on its ability to manipulate the CYP transcriptome, therefore improved understanding of how key mutations alter CYP gene splicing outcomes may be critical to addressing the goal of precision medicine in the post-genomic era.

TISSUE-SPECIFIC ALTERNATIVE SPLICING OF CYTOCHROME P450 GENES: A

HISTORICAL PERSPECTIVE

During the late 1980’s and early 1990’s, several groups began to appreciate the possibility that differential RNA splicing might serve as an additional mode for regulating CYP function. In

1987, the first P450 splice variant for hepatic P450-1, later renamed CYP2C8, was reported

(Okino et al., 1987). Over the next decade, several examples of alternate splicing were

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 documented for several human CYPs including lung CYP4F2 (Nhamburo et al., 1990), liver

CYP2B6 (Yamano et al., 1989; Miles et al., 1989) and CYP2A7 (Ding et al., 1995). In the latter case, a tissue-specific, alternative splicing effect was documented, where a 44 kDa CYP2A7-AS splice variant (compared to 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, Downloaded from 1997). Circular RNA transcripts synthesized from the donor and acceptor sites from the skipped segments of exons 4-7 were also documented, and although their potential epigenetic functions

remains unknown, they were easily detected based on their increased stability (Zaphiropoulos, dmd.aspetjournals.org

1997). Important studies focused on environmentally-responsive, alternative CYP splicing among xenobiotic-metabolizing P450s of the rat 2A family were also conducted during this era

(Desrochers et al. 1996). at ASPET Journals on September 30, 2021 By 1999, the CYP2C18 alternative splicing story became even more complex when it was discovered that CYP2C18 and CYP2C19, which cluster on chromosome 10q24 with CYP2C9, also participate in tissue-specific, trans-splicing events, among the 3 genes (Zaphiropoulos,

1999). Over the next decade, cell-specific splicing factors responsible for directing the complex, post-transcriptional gene assembly of P450s (and other genes) were identified (Wang and Burge,

2008). However, the physiological importance of the more unusual CYP splice variant and chimeric forms remains unclear, particularly with respect to how their variable, tissue-specific expression alters inter-individual sensitivity to hormones, pharmaceuticals and xenobiotics.

As discussed above, a single CYP gene can be translated into multiple, variant protein sequences, based only on tissue-specific differences in the expression of key, gene splicing machinery (e.g. snRNAs). Several alternately-spliced CYP1A1 transcripts identified in human brain tissues exemplify this phenomenon, as well as the concept of tissue-specific spliceosome activity (Bauer et al., 2007). Inducible CYP1A1 is constitutively expressed in the brain, where it

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 is localized to neurons in the cortex, cerebellum and hippocampus. An 87 bp deletion in exon 6 is observed in the brain-specific CYP1A1 form, but not in liver (Chinta et al., 2005). Human brain tissues expressing the Δexon6-CYP1A1 variant do not express wild-type CYP1A1 and they metabolize known substrates (e.g. benzoxy-resorufin and benzo(a)pyrene (BP)) at different rates than wild-type enzyme (Kommaddi et al., 2007). Furthermore, cells expressing wild-type CYP1A1 generate DNA adducts via the formation of reactive 3-OH-BP products, while cells expressing the

Δexon6-CYP1A1 variant in brain, do not (Kommaddi et al., 2007). Downloaded from Based on the structural analysis of CYP1A1 in Figure 2 (Derived from PDB: 4I8V; Walsh et al., 2013), tissue-specific, alternative splicing of exon 6 may re-shape the size and polarity of

the CYP1A1 substrate access channel, to alter substrate recruitment and recognition processes dmd.aspetjournals.org and redox-partner binding interactions. Exon 6 of CYP1A1 appears to be organized for “plug and play” utilization in the brain, where targeted skipping does not compromise the global P450 fold, but alters substrate specificity for a spectrum of endogenous substrates, including at ASPET Journals on September 30, 2021 testosterone. This tissue-specific, exon 6 usage of CYP1A1 is complimented by a similar pattern of alternative exon 2 usage found in human endothelial cells, leukocytes and tumor cells (Leung et al., 2005, Bauer et al., 2007). As shown in Figure 2, targeted-skipping of an 84 bp cryptic intron in exon 2 of CYP1A1, produces catalytically-unique, splice variant with diminished estradiol metabolism that localizes to the nucleus and mitochondria rather than the ER (Leung et al., 2005).

The added diversity in both structure/function and subcellular distribution observed for exon 2- modified, CYP1A1 splice variants found both in normal and tumor tissues remains enigmatic and denotes a higher level of structural plasticity with respect to tertiary structure, than might be currently appreciated or expected from classical structural studies of wild-type CYP forms

(Johnson and Stout, 2013).

Perhaps the most dramatic, and mutually exclusive example of tissue-specific, alternate exon usage in P450s was documented for CYP4F3, which can be expressed as two, distinct splice variant forms (CYP4F3A and CYP4F3B). These two CYP4F3 isoforms differ only by their

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 variable usage of exons 3 and 4, which encode distinct portions of the active site and substrate access channel, allowing them to fine-tune substrate specificity (Christmas et al., 1999; Christmas et al., 2001). CYP4F3A is expressed in blood and bone marrow myeloid cells, selectively expresses cassette exon 4 (and not 3), and has low affinity for leukotriene B(4) (LTB4). In contrast, CYP4F3B, expressed exclusively in the liver and kidney, encodes exon 3 (and not 4) and has high affinity for LTB4, in addition to other functional differences. The highly concerted, tissue-specific splicing of CYP4F3 exemplifies the functional utility of the modular CYP gene Downloaded from platform, one that allows cells to sample a continuum of a gene products, as dictated by tissue- specific, physiological stress conditions.

However, it should also be clearly noted, that not all CYP genes are divided into an equal dmd.aspetjournals.org number of coding and non-coding regions, which suggests that the accumulation of introns may be an adaptive and ongoing process. For example, the CYP1B1 gene, which encodes a P450 enzyme that metabolizes both xenobiotics and endogenous substrates, contains only 2 coding at ASPET Journals on September 30, 2021 exons (Supplemental Figure 2), and therefore is not subject to the same patterns of alternative splicing as other CYP1 family members, 1A1 & 1A2, which have 6 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 CYP forms. The

CYP1B1 gene is also uniquely located on chromosome 2 at locus 2p21-22, which in humans, is derived from an ancient fusion of chimpanzee chromosomes 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 more related to the rate at which a given mRNA transcripts are processed by the spliceosome and ribosome, rather than other evolutionary factors (Lejeune and Maquat, 2005;

McGlincey and Smith, 2008). Ultimately, the adaptive forces driving the modular organization of

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 genes into variable numbers of introns and exons remain a point of speculation. However, a gene’s intronic complexity clearly influences its sensitivity to alternate splicing events and the domain swapping paradigm. The tissue-specific use of alternative promoters and alternative

(AUG) translational-start-sites also is facilitated by this type of hidden gene complexity that allows discrimination among a set of well-defined exons and poorly-defined pseudoexons.

In 2005, a truncated, CYP24A1 splice variant was identified in the human macrophage that is incapable of metabolizing hormonal forms of vitamin D (Ren et al., 2005). CYP24A1 is the Downloaded from prototypical mitochondrial P450 responsible for the side-chain cleavage of the vitamin D hormone and it possesses a mitochondrial localization sequence in the first 30 residues of its N-terminus

(Annalora et al; 2004). The CYP24A1 splice variant found in activated macrophage (CYP24A1- dmd.aspetjournals.org

SV1) lacks 153 N-terminal residues encoded by exons 1 and 2, and gains 8 residues preceding the content from exon 3, via a partial insertion of intron 2. CYP24A1-SV1 is thought to function as a soluble variant that disrupts normal vitamin D hormone metabolism via the sequestration of at ASPET Journals on September 30, 2021 key intermediates from both wild type CYP27B1 and CYP24A1 (Ren et al., 2005). In this regard, the CYP24A1-SV1 variant may promote a discrete pattern of vitamin D hormone accumulation in the macrophage, and potentially other tissues, such as the intestine, where 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 structural analysis of the CYP24A1-SV1 variant is shown in Figure 3 (based on PDB: 3K9V;

Annalora et al., 2010), and it highlights how the targeted loss of N-terminal residues from exons

1 and 2 could reconfigure the membrane-binding surface and substrate access channel to alter the catalytic properties of wild-type CYP24A1.

Interestingly, the orphan P450 CYP27C1, a close relative of CYP24A1, encodes a 372 residue wild-type protein that resembles the CYP24A1-SV1. CYP27C1 lacks a prototypical N- terminus and initiates coding in the middle of canonical helix C, at a position analogous to the alternative start site utilized by CYP24A1-SV1 (Ren et al., 2005). CYP27C1 is expressed in liver,

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 kidney, pancreas and several other tissues, and remained an orphan for over a decade (Wu et al., 2006), until recently, when it was shown to mediate the conversion of vitamin A1 (all-trans retinol) into vitamin A2 (all-trans 3,4-dehydroretinal) in cell culture (Kramlinger et al., 2016). It is intriguing that CYP27C1 may represent a codified version of the more soluble CYP isotype recapitulated by CYP24-SV1, and that this transition may mediate a general switch between vitamin A and vitamin D substrate specificity. Improved knowledge of CYP27C1 structure/function could help to unravel some of the complexity associated with the variant CYP regulation, Downloaded from particularly for variants subject to atypical sub-cellular trafficking events.

Therefore, while increasingly common in the literature, the functional significance of many

tissue-specific CYP variants remain elusive. CYP2C8 for example, which is constitutively dmd.aspetjournals.org expressed in the liver at 5-7% of the total tissue P450, has more than 20 known alternately-spliced variants, and is a breast cancer biomarker due to its high expression in mammary tumors (Knüpfer et al., 2004). Bimodal targeting to both mitochondria and ER have been reported for CYP2C8 at ASPET Journals on September 30, 2021 and several other CYPs, including CYP1A1, 1B1, 2B1, 2E1 and 2D6. While it is clear that alternative splicing directs this process in some tissues, proteolytic cleavage of N-terminal targeting sequences can also produce this effect (Bajpai et al., 2014). One 2C8 variant (variant

3; ~44 kDa) with an alternative N-terminus is targeted to the mitochondria where it may contribute to oxidative stress (Bajpai et al., 2014). The catalytic competency of these untethered variants remains a topic of intense debate, as it is often difficult to assign an orphan function to a variant protein that has lost selectivity to the gene’s prototypical substrates. A review of CYP splice variants, subject to alternate subcellular trafficking is shown in Supplemental Table 1.

Computational analysis from the Aceview database (Thierry-Mieg and Thierry-Mieg, 2006) indicate that some CYP variants may be targeted to the nucleus, peroxisome, plasma membrane or vacuole compartments, in addition to classical targets in the ER, mitochondria or cytoplasm.

This analysis hints that CYP splice variants, including tumor-specific forms, may be performing

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 an array of unrecognized functions, including diverse roles at the cell surface and beyond, which are rarely considered (Eliasson et al., 1996; Stenstedt et al., 2012).

TRANSFORMATION-SENSITIVE ALTERNATIVE SPLICING IN THE CYTOCHROME P450

SUPERFAMILY

This transformation-specific, CYP splicing phenomenon was first documented in 1996 for the prototype P450, CYP19A1 (or aromatase), which facilitates estrogen biosynthesis via three Downloaded from successive hydroxylations of the androgen A ring and displays a unique pattern of exon 1 (and cryptic intron) usage in human breast tumor cells and tissues (Zhou et al., 1996). A similar pattern

of CYP19A1 exon 1 variant expression also has been documented in sheep, cattle and rabbit, dmd.aspetjournals.org suggesting this paradigm is not exclusive to humans or transformed-tissue types (Vanselow et al., 1999; Bouraïma et al., 2001). More recently, it was determined that CYP19A1’s exon 5 also is defined poorly and subject to both splicing mutations and physiological alternative splicing at ASPET Journals on September 30, 2021 events (Pepe et al., 2007). Skipping of exon 5, 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). These observations suggest a tissue- specific, regulatory mechanism for aromatization based on alternative exon 5 inclusion in humans.

A structural analysis of this CYP19A1 splice variant is depicted in Supplemental Figure 3 (Based on PDB: 3S79; Ghosh et al., 2012). In aromatase, exon 5 skipping naturally suppresses androgen metabolism by re-defining the determinants of substrate recognition. While transfection studies suggest CYP19A1 variant proteins are encoded and not subject to NMD, their ability to alter the safety and efficacy of several CYP19A1 (aromatase) inhibitor drugs (e.g. AIs; including Arimidex

(anastrozole); Aromasin (exemestane); Femara (letrozole) and Teslac (testolactone) remains unclear (Hadfield and Newman, 2012) and an area of active investigation (Liu et al., 2016).

The tissue-specific, alternative start site usage of CYP24A1, discussed above, appears to be exacerbated in several human transcript variants, where an array of truncated splice variants

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 of approximately 40, 42, and 44 kDa have been identified with similar N-terminal modifications

(Fischer et al., 2009; Horváth et al., 2010; Scheible et al., 2014). An additional, cancer-related, splice variant of CYP24A1 that cleanly skips exon 10 has also been documented (Horvath et al.,

2010; Muindi et al., 2007). In CYP24A1, exon 10 encodes much of the protein’s proximal surface, including the CYS loop, and a portion of helix L involved in heme-thiolate bond formation and adrenodoxin recognition (shown in Figure 3). While the function of this cancer-specific variant remains unknown, it may have reduced ability to bind heme and redox partners compared to Downloaded from truncated variants (skipping exons 1 and 2), and thus may represent an alternative, dominant negative variant also capable of modulating vitamin D hormone metabolism and metabolite

trafficking at the plasma membrane level. As discussed previously, the cellular mechanisms dmd.aspetjournals.org regulating CYP24A1 alternative spicing in the macrophage remains unclear, although cell-specific variations in splicing factors (e.g. heterogeneous ribonuclear protein A1) are thought to be responsible (Ren et al., 2005). A more complex pattern of “transformation-sensitive’ alternative at ASPET Journals on September 30, 2021 CYP24A1 gene splicing also has been documented in human tumors of the prostate (Muindi et al., 2007), breast (Fischer et al., 2009; Scheible et al., 2014) and colon (Horváth et al., 2010; Peng et al., 2012). It is thought that while intronic SNPs may facilitate the alternate splicing pattern seen for CYP24A1 in prostate cancer cells, vitamin D hormone exposure also putatively alters splicing via discrete interactions among the vitamin D receptor (VDR) and nuclear splicing factors like the heterogeneous nuclear ribonucleoprotein C (hnRNPC; Zhou et al; 2014). Tumor cells overexpressing CYP24A1, therefore, may not retain sufficient hormone to activate the VDR properly, disrupting normal gene splicing events needed to properly transduce the hormone’s immunogenic, pro-differentiation properties. CYP24A1 splice variants have now been documented among multiple human breast tumor cell lines (e.g. MCF-7 and MCF-10) and in both healthy and malignant breast tumor tissues. In benign tissue, wild-type CYP24A1 (56 kDa) was the only isoform present. In malignant tissues, 3 splice variants (40, 42 and 44 kDa) were expressed, similar to cultured MCF-7 breast tumor cells that express the 42 and 44 kDa variants

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

(Fischer et al., 2009; Scheible et al., 2014). A similar pattern of transformation-sensitive splicing is seen in human colon cancer tissues, where the histological grade and the gender of the patient modulates the formation of the 3 detectable CYP24A1 splice variants (Horváth et al., 2010). Peng and co-authors further refined our understanding of this process in the colon, by demonstrating that cell- or tissue-specific splicing patterns were suppressed by parathyroid hormone signaling, but only in the absence of the vitamin D hormone, which may be the master regulator of both

CYP24A1 gene expression and splicing (Peng et al., 2012). Downloaded from The phenotypic instability exemplified by the CYP24A1 gene in human tumors complicates the therapeutic landscape from which rational drugs for cancer can be conceived (Trump et al.,

2006). Epigenetic modification of the CYP24A1 gene and its promoter also may contribute to dmd.aspetjournals.org tissue-specific differences in P450 expression (Johnson et al., 2010). Frontiers in this area of research will likely address the coordinated role that nuclear hormone receptors and epigenetic splicing factors (including non-coding forms of RNA) play in managing both the transcriptome and at ASPET Journals on September 30, 2021 the epigenome.

It should be noted that the CYP27B1 gene, which is highly related to CYP24A1, was also recognized early as being sensitive to transformation-specific alternative splicing in malignant human glioma cells (Maas et al., 2001). CYP27B1 variant expression has now been documented in tumors of the breast, skin, cervix, kidney and ovaries (Fischer et al., 2007; Wu et al., 2007;

Diesel et al., 2004; Fischer et al., 2009). Tissue-specific CYP27B1 splicing also is common in healthy skin, where a 59 kDa variant with a 3 kDa insertion between exons 2 and 3, exclusive to skin, was observed, and linked to the activity of CRE-binding elements (Flanagan et al., 2003).

Fluctuations in skin cell density, calcium concentrations, and UV-B light exposure were also shown to effect the splicing process (Seifert et al., 2009). Collectively, these findings help to validate our core hypothesis that alternative gene splicing, dictated by both cellular and environmental factors such as UV light exposure and nutritional status, can alter a patient’s

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 metabolic profile in tissue-, age-, sex-, exposure- and disease-specific manners that are difficult to predict.

While research focused on CYPs 19A1, 24A1 and 27B1 are among the most comprehensive studies on transformation-sensitive alternative splicing in human P450s, the literature is replete with anecdotal reports of alternative CYP splicing among various transformed cell-types and tissues. For example, CYP2E1 transcripts that selectively skip exons (2, 2-3, and

2,4,5-5’,6) were identified in lung carcinoma cell lines, but not in corresponding lung tumor tissue Downloaded from or liver extracts, where splice variants were found to be non-inducible by ethanol exposure (Bauer et al., 2005). The authors noted at the time that none of these splice variants were accounted for

in the NIH’s Aceview database, highlighting the longstanding challenge associated with cataloging dmd.aspetjournals.org splice variant forms derived from cell–specific abnormalities versus those generated from normal, tissue-specific splicing paradigms. Furthermore, several orphan P450s, including CYP1B1,

CYP2S1 and CYP2W1 are highly associated with specific, human tumor types, making them at ASPET Journals on September 30, 2021 important pro-drug targets for chemoprevention strategies (Stenstedt et al., 2012). Unfortunately, as discussed above, the alternative splicing behavior of orphan CYPs is often as poorly understood, as their cryptic function.

Interestingly, orphan CYP2W1, which is expressed human tumor types of the colon, is associated almost exclusively with aberrant P450 behavior, including an inverted orientation in the ER membrane, and glycosylation-sensitive trafficking to the plasma membrane (Gomez et al.,

2010; Stenstedt et al., 2012). The increased expression of CYP2W1 in tumors is associated with demethylation of a CpG island in the exon 1-intron 1 junction of the gene. How this mechanism relates to the paucity of normal CYP2W1 expression in healthy tissues, remains unclear, but it implies that the expression of some specialized CYPs may only be invoked under extreme cellular stress, where an alternative CYP activity may be critical for balancing cellular homeostasis. The abnormal expression and trafficking of CYP2W1 to the plasma membrane could promote an autoimmune response as part of its mechanism. A similar autoimmune reaction is associated

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 with aberrant CYP2E1 trafficking in animal tissues treated with halothane (Eliasson et al., 1996).

The NIH’s Aceview and Ensembl (Cunningham et al., 2015) databases indicate CYP2W1 encodes at least 11 transcript variants, three of which lack one or more exons and are associated with hepatocellular carcinomas, adenocarcinomas and neuroblastomas. Nearly 200 of the 965 alternatively-spliced CYP transcripts we identified in public database searches were associated with one of 40 human tumor types. Improved understanding of their tumor-specific cellular roles, and the mechanisms regulating their induction, should aid in the identification of improved disease Downloaded from biomarkers and CYP variant-specific therapeutic agents.

TRANS-SPLICING OF CYTOCHROME P450 GENE CHIMERAS dmd.aspetjournals.org

It is well understood that both transformation-sensitive cellular changes and CYP polymorphisms can alter protein expression in multiple ways that are often difficult to predict from primary sequence analysis alone. This challenge is exacerbated in situations where chimeric at ASPET Journals on September 30, 2021 CYP genes are created via trans-splicing events among extended (or multiple) pre-mRNA transcripts. Therefore, while mutations in a gene like CYP2C9 are linked to poor metabolism of several drugs, including phenytoin and tolbutamide, the role that alternative- or trans- gene splicing plays in manifesting a deleterious CYP2C9-related metabolic phenotype are rarely considered. For example, several CYP2C9 splice variants (CYP2C9sv) have been identified

(Ohgiya et al., 1992), including a liver-specific form that skips exon 2 (Ariyoshi et al., 2007). The internally-truncated CYP2C9 variant is spectrally-active, but does not metabolize prototype

CYP2C9 substrates. A structural analysis of the CYP2C9 crystal structure (PDB: 4NZ2; Brändén et al., 2014) shown in Supplemental Figure 4A, hint that removal of exon 2 would reshape portions of the membrane-binding surface, substrate access channel, active site and redox partner binding surface relative to the reference protein. CYP2C9 is located within a cluster of CYP genes on chromosome 10q24 and is subject to non-random, trans-splicing events with neighboring CYPs

2C8, 2C18 and 2C19 in liver and skin, where CYP2C18 exon 1–like sequences were found

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 spliced into different combinations of introns and exons from the CYP2C9 genes (Warner et al.,

2001). Common 2C9 related SNPs linked to various metabolic disorders, therefore, may only function to modulate the innate splicing process, indirectly. This is particularly true if their coding regions are spliced out of chimeric 2C forms being expressed on an inter-individual basis, highlighting another major flaw in GWAS targeting the components of gene clusters.

The CYP2D gene family also is complex, clustered and subject to trans-splicing events; it is comprised of CYP2D6 and 4 pseudogenes (CYP2D7P1 and 2 and CYP2D8P1 and 2). Downloaded from Alternative splicing of the CYP2D family pre-mRNAs has been detected in human liver, breast and lung tissue (Huang et al., 1996; Huang et al., 1997), including a highly-expressed splice

variant which completely skips exon 6. As shown in Supplemental Figure 4B, analysis of the dmd.aspetjournals.org

CYP2D6 crystal structure (PDB: 4WNU: Wang et al., 2015) reveals that exon 6 encodes the complete coding sequence for helix I, and that skipping of this important region would dramatically alter protein structure/function, by removing key catalytic residues (e.g. Thr-309) and remodeling at ASPET Journals on September 30, 2021 the distal pocket of the active site. Alternate CYP2D6 exon usage is tissue-selective and variant forms skipping exon 3 and portions of exon 4 have been documented that alter enzyme function and subcellular localization (Sangar et al., 2010). CYP2D6 polymorphisms are linked to modified enzyme activity and may serve as biomarkers for poor metabolizing phenotypes sensitive to dose- dependent drug toxicity. Recently it was reported that intronic polymorphisms in CYP2D6*41 individuals (considered intermediate metabolizers) could dramatically increase the expression levels of CYP2D6 variants lacking exon 6 (Toscano et al., 2006). These findings, coupled with the complex organization of the CYP2D gene cluster, indicate that both alternative and trans- splicing mechanisms may underlie the complex genotype-phenotype relationships associated with variable CYP2D6 metabolism in humans, and that CYP2D6 is well-organized structurally to exploit both tissue- and transformation-selective splicing mechanisms targeting helices C and D

(exon 3) and helix I (exon 6).

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

The CYP 3A gene family (comprised of CYP 3A4, 3A5, 3A7 and 3A43) is also highly complex in humans, with each gene consisting of 13 exons (with ~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 mRNAs have been described where CYP3A43 exon 1 is joined at CYP3A4 and CYP3A5 canonical splice sites, implying a trans-splicing mechanism that bypasses classical transcriptional control paradigms. In Supplemental Figure 5A, a structural analysis of CYP3A4 (Based on PDB: Downloaded from 4K9T; Sevrioukova et al., 2013) reveals the highly-complex segmentation of the CYP3A4 gene into 13 exonic domains, which, as described above, may potentiate rapid translation upon

induction (Lejeune and Maquat, 2005; McGlincy and Smith, 2008). Primary amino acid sequence dmd.aspetjournals.org alignments of key CYP3A genes, suggest exon 1 may encode distinct transmembrane anchor helices and membrane binding architecture (Supplemental Figure 5B), hinting that CYP3A chimeras may encode a spectrum phenotypes with unique tissue-specific distribution and at ASPET Journals on September 30, 2021 functionalities. A CYP3A4 trans-splicing variant containing CYP3A43 exon 1, followed by

CYP3A4 exons 4-13, has been described, which retains detectable, testosterone 6-β-hydroxylase activity despite losing membrane binding features encoded by exons 2 and 3, comprising helices

A’ and A, and portions of the β1-1 sheet (Supplemental Figure 5C). A CYP3A4 trans-splicing variant containing CYP3A43 exon 1, followed by CYP3A4 exons 7-13 also has been studied, and it displays minimal 6-β-hydroxylase activity (Supplemental Figure 5D). This story is further complicated by the presence of additional CYP3A pseudogenes (CYP3AP1 and CYP3AP2) in the intergenic regions between CYP3A4, 3A7 and 3A5, where discrete exons from the pseudogenes also have been found trans-spliced into CYP3A7 transcripts (Finta and

Zaphiropoulos, 2002). Ultimately, CYP3A4 and CYP3A5 splicing complexity may help to explain how intronic SNPs in CYP3A4*22 patients alter tacrolimus metabolism, without introducing a non- synonymous mutation to an exon (Elens et al., 2011). The predictive power of GWAS may therefore need to be reconsidered, particularly with respect to the most complex CYP gene

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 families defined by the presence of pseudogenes (e.g. CYP 2C, 2D, 3A, 21A and 51A families) and trans-splicing mechanisms.

SNP-SENSITIVE ALTERNATIVE SPLICING IN THE CYTOCHROME P450 SUPERFAMILY

As discussed above, tissue-specific, alternative- and trans-splicing behavior has now been documented for several, important CYP gene families, however single nucleotide polymorphisms

(SNPs) can destabilize this process in complex ways that are often difficult to predict. SNPs that Downloaded from target cryptic intronic recognition elements or discrete intron/exon splice junction boundaries can alter translation start and stop site usage and the expression ratios among wild-type and variant

CYP transcripts. Some CYP SNPs are exceedingly rare, occurring in less than 1% of the dmd.aspetjournals.org population. These rare mutations are linked to congenital defects, including glaucoma (CYP1B1;

Stoilov et al., 1998; Tanwar et al., 2009; Sheikh et al., 2014), 17-alpha hydroxylase deficiency

(CYP17A1; Yamaguchi et al., 1998, Costa-Santos et al., 2004; Hwang et al., 2011; Qiao et al at ASPET Journals on September 30, 2021 2011), congenital adrenal hyperplasia (CYP21A2; Robins et al., 2006; Szabó et al., 2013, Lee,

2013; Sharaf et al., 2014), spina bifida (CYP26A1; Rat et al., 2006), focal facial dermal dysplasia

(CYP26C1; Slavotinek et al., 2013), and cerebrotendinous xanthomatosis (CYP27A1; Garuti et al., 1996; Verrips et al., 1997; Chen et al., 1998; Tian et al., 2011). Other CYP polymorphisms that alter splicing are associated with neurological and metabolic diseases, including Parkinson’s disease (CYP2D6; Denson, 2005; CYP2J2; Searles et al., 2013), hypertension (CYP4A11; Zhang et al., 2013; CYP17A1; Wang et al., 2011), breast cancer (CYP2D6; Huang et al., 1996;

CYP19A1; Kristensen et al., 2000;) colon cancer (CYP2W1; Stenstedt et al., 2012) and lung cancer (CYP2D6; Huang et al., 1997; CYP2F1; Tournel et al., 2007). However, the complex etiology of these disorders can make it difficult to interpret the exact role that a P450 SNP might play in the actual onset or progression of a disease. Multiple studies of various intergenic, intronic and exonic polymorphisms that alter human drug and xenobiotic metabolism have also been reported (CYP1A1; Allorge et al., 2003; CYP2C19; deMorais et al., 1994, Ibeanu et al., 1999,

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Satyanarayana et al., 2009, Sun et al., 2014; CYP2D6; Toscano et al., 2006, Lu et al., 2013,

Wang et al., 2014; CYP3A4; Elens et al., 2011; and CYP3A5; Kuehl et al., 2001, Busi and

Cresteil, 2005, Lee et al., 2007, García-Roca et al., 2012). In many cases, the most pharmacologically-relevant SNPs are those affecting the innate splicing behavior of a given gene.

This observation highlights why knowledge of a patient’s discrete CYP transcriptome is so important when selecting a personalized medicine regime.

It is now clear that numerous CYP-related SNPs are found between genes and exons, or Downloaded from in the 5’ or 3’ untranslated regions of RNA transcripts. Polymorphisms in this class have now been identified for virtually every human CYP isoform, with each having the potential to alter

splicing in unique ways in different individuals. For example, the CYP3A5*3 SNP (rs776746, dmd.aspetjournals.org also known as 6986A>G) introduces a mutation in intron 3 that alters normal splicing of exon 4, producing a nonfunctional, truncated CYP3A5 protein that predominates in many Caucasians

(Kuehl et al., 2001). Expression of the wild-type CYP3A5*1 protein is highly-variable among at ASPET Journals on September 30, 2021 populations, and is linked to an increased sensitivity to salt-induced hypertension. Interestingly, some individuals that are homozygous CYP3A5*3/*3 can express both the truncated variant and properly-spliced, wild-type CYP3A5 (Lin et al., 2002). Because the CYP3A5*3 mutation occurs in intron 3, over 100 base pairs upstream from the splice donor site of exon 4, it is unclear how this SNP alters normal recognition of the intron 3-exon 4 junction. While variations in the expression of ubiquitous splicing factors (e.g. U1snRNP) may account for inter-individual variability in recognizing 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 exemplifies why SNP-based approaches to personalized medicine are so challenging to implement, when the physiological impact of a given SNP, may be the opposite of what is expected. Computational approaches that consider a patient’s age, genotype, nutrition and disease status, may ultimately be needed to make good predictions concerning the impact of a given SNP on an individual’s transcriptome.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

The challenge of addressing SNPs for personalized medicine is further complicated by observations that some xenobiotics, including aminoglycosides and cyclohexamides (Busi and

Cresteil, 2005), can alter the proofreading capabilities of the ribosome, allowing inhibition of PTCs and translational read-thru of alternate transcripts that may or may not remain in-frame with respect to the wild-type transcript. Improved knowledge of a patient’s chemical exposure history and epigenetic status, therefore, may become increasingly important when making predictions about food and drug safety. Furthermore, personalized approaches to medicine, based solely on Downloaded from SNPs identified via cohort studies or GWAS, may be fundamentally-flawed, and potentially misleading, in that they fail to account for the milieu of compensatory cellular mechanisms that

are adept at masking the most deleterious effects of even the most highly-penetrant mutations. dmd.aspetjournals.org

ADDRESSING NON-TRADITIONAL CYP FUNCTION AND TRAFFICKING EVENTS

Our meta-analysis and review of splice variability within the cytochrome P450 superfamily at ASPET Journals on September 30, 2021 has revealed an under-appreciated level of alternative transcript expansion and variant protein structural complexity that is poorly addressed by conventional CYP structure/function paradigms.

Well-studied splice variants from the CYP1A family reveal a sophisticated domain swapping mechanism that allows for tissue-specific sampling of alternative protein conformational space.

Discrete arrays of tissue-specific snRNAs (U1-U9) and ancillary splicing factors can alter the biological syntax of a gene, thus providing a granular mechanism for this natural phenomenon, which is highly-responsive to environmental factors, including diet and chemical exposure. In human brain, the alternative splicing of CYP1A1 appears linked to a tissue-specific sensitivity to a reactive CYP-product (e.g. 3-OH BP; Kommaddi et al., 2007). How this gene-specific sensitivity is transduced and imprinted at the genetic level remains enigmatic, but the cellular microenvironment clearly dictates what hidden structural complexity of a CYP gene may be exploited. This observation helps explain why some CYP variants, like CYP24A1-SV1, which are normally expressed in the immune system, can become major players in tumor cells, where they

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 may elaborate a common emergency function, such as the suppression of vitamin D hormone metabolism. This cellular phenomenon corresponds well with related, vitamin D hormone- scavenging strategies, such as the 3-epimerization pathway, which can be induced to modify the vitamin D hormone’s chemical ring structure to limit its efficient catabolism via CYP24A1 (Rhieu et al., 2013). To date the enzyme responsible for the 3-epimerization of the vitamin D hormone remains unknown (Bailey et al., 2013).

New appreciation for the increasingly diverse and complex roles that CYP splice variants Downloaded from play in normal biology should help reinvigorate discussions of CYP gene structure and evolution, particularly with respect to the selective forces that regulate CYP gene duplication and mutation

events. While the origin and expansion of spliceosomal introns remains highly-debated (Rogozin dmd.aspetjournals.org et al., 2012; Qu et al., 2014), the intron-exon boundaries of 17 of 18 mammalian CYP families appear to be well-conserved across almost 420 million years of evolution, as evidenced by the fugu (pufferfish) genome (Nelson, 2003); only the CYP39 gene is missing from fugu and all known at ASPET Journals on September 30, 2021 fish. Th bulk of P450 structural diversity encoded by the human genome, therefore, likely predates the divergence of tetrapods from ray-finned fish over 420 million years ago. In this regard, brain- selective expression of CYP1A1 variants lacking exon 6 in humans, may be no more evolved than tissue-specific expression of CYP19A1 splice variants found in fugu. In each case, the induction of CYP splice variants appears to provide a novel mechanism for modulating steroid hormone pleiotropism among a diverse set of target tissues.

The selective pressures that created the modular framework of the CYP gene, therefore likely predate the Cambrian explosion 540 million years ago, and lurk somewhere in the history of deuterostome evolution (Nelson, 2003). However, contemporary analysis of CYP splicing mechanisms are needed to help elaborate the relationship between alternative subcellular trafficking and alternative CYP variant function in the mitochondria, nucleus, peroxisome, cytoplasm or plasma membrane (Supplemental Table 2). Because alternative splicing and alternative start site usage can generate even greater CYP structural diversity than those

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 predicted to arise from post-translational modifications of intracellular targeting motifs, the mechanisms underlying their emergence and regulation deserve greater attention. For example, if subtle differences in the membrane binding features of CYP1A1 and CYP1A2 can modulate redox partner recruitment and protein interactions within ordered and disordered regions of the lipid bilayer (Park et al; 2015), metamorphic structural changes associated with some CYP variants may underlie an even greater array of uncharacterized (and potentially moonlighting)

P450 functions (Zhao and Waterman 2011, Lamb and Waterman, 2013). Downloaded from While the landscape of alternate CYP gene functions continues to expand, the cellular basis for alternatively-targeting microsomal CYP forms to the mitochondria (e.g. CYP1A1,

CYP1A2, CYP1B1 CYP2B1, CYP2B2, CYP2E1, CYP3A1 and CYP3A2) is still being investigated dmd.aspetjournals.org

(Anandatheerthavarada et al, 1997). However, a putative role in modulating destructive, subcellular ROS production has recently been proposed (Dong et al., 2013). This theory emerges from evidence that the alternative trafficking of the related NADPH:quinone oxidase-1 (NQO1) at ASPET Journals on September 30, 2021 gene, which limits ROS by preventing semiquinone redox cycling, can protect the mitochondria from stress-induced oxidative damage. CYP variants may potentially play a similar role by redirecting the metabolism of reactive subcellular compounds. However, because CYPs can also become uncoupled and directly produce ROS via the release of reactive products (i.e. via uncoupled Fenton-type reactions), further attention to the role of alternative CYP splicing in the modulation of mitochondrial stress and dysfunction seem warranted. In this regard, it is intriguing that many CYP splice variants that undergo alternative trafficking often lack a significant portion of the membrane binding domain and/or substrate access architecture (typically encoded by exons 1-3), which may expand heme accessibility or the rate of uncoupled ROS production.

When paired with observations that alternative splicing is a primary mechanism by which mitochondria alter cellular phenotypes to mitigate environmental stress (Guantes et al., 2015), new, non-classical roles for CYP variants linked to disease, seem likely to emerge.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

In this regard, it is interesting that the consensus sequence (GU-AG) of most RNA splice junctions contains a minimum of two guanine nucleotides, which are highly sensitive to ROS- mediated oxidation. In the presence of radical oxygen, guanine is rapidly converted to 8-oxo- guanine and related metabolites that disrupt normal base pairing and local secondary structure.

Formation of 8-oxo-guanine at splice junctions can impede target site recognition by splicing factors (e.g. snRNAs and SRF proteins) and alter RNA Pol II processivity. Furthermore, because some introns are defined by AU-AC junctions rather than GU-AG junctions, the splicing of these Downloaded from introns may be less sensitive to ROS-mediated events, and therefore may be subject to a different suite of regulatory splicing mechanisms. For example, the induction of an environmentally-

responsive genes, like adenosine-deaminase could alter the recognition of a prototypical AU-AC dmd.aspetjournals.org junction in a similar way, via adenosine-to-inosine (A-to-I) RNA editing (Solomon et al., 2013).

Even mitochondrial genes lacking spliceosomal introns could be affected by this phenomenon, as the discrete dinucleotide sequences that coordinate their autocatalytic, self-splicing events, at ASPET Journals on September 30, 2021 could also be potentially altered by CYP-mediated ROS modulation (Zimmerly and Semper,

2015).

SHAPING AN IMPROVED ROADMAP TOWARDS PRECISION MEDICINE

Because changes in mitochondrial function can alter global alternative splicing events, it is not surprising that both phenomenon are linked to the induction of cellular heterogeneity and human disease pathology progression (Raj and van Oudenaarden, 2008; Hanahan and

Weinberg, 2012; Pagliarini et al., 2015). Despite a growing appreciation for the role that alternative splicing plays in promoting phenotypic variability, the identification of genetic polymorphisms linked to disease, which may or may not alter gene splicing events, remain a core focus of Pharmacogenomics. Mutations in single genes have now been identified for over 4000 human diseases, of which, 5 to 15 % are the result of SNPs resulting in nonsense mutations (Mort et al., 2008). However, SNPs occur in coding regions of genes with a frequency of approximately

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

1.5 %, and of those, only one third are expected to result in non-synonymous mutations and a much smaller number alter pre-mRNA processing, leading to a frequency in change of phenotype only ~0.5 % of the time. When one considers humans express almost 50,000 SNPs across the

57 human CYP genes, fewer than 250 may capable of significantly altering a patient’s metabolic phenotype substantially, an insight we contend may help streamline PGx approaches to personalized medicine (Zhou et al., 2009; Zanger and Schwab, 2013). For example, while

CYP2D6 genotyping is no longer recommended in the clinical setting for Tamoxifen treatments Downloaded from (Abraham et al., 2010; Wu, 2011, Lum et al., 2013), the FDA still considers CYP2D6 clinically actionable from a PGx perspective for codeine and other drugs (Crews et al., 2014).

Unfortunately, over 74 allelic variants of CYP2D6 have already been described, which greatly dmd.aspetjournals.org complicates genetic testing and the clinician’s ability to select the appropriate pharmacotherapy and dose (Zhou, 2009). Fortunately, organizations such as the Clinical Pharmacogenetics

Implementation Consortium (CPIC) the Pharmacogenomics Knowledgebase (PharmGKB) are at ASPET Journals on September 30, 2021 working to standardize methodologies for PGx data analysis and clinical guidance for specific gene-drug pairings (Caudle et al., 2016). However, CPIC concedes that some rare CYP variants may not be included in genetic tests, and that patients may be assigned “wild-type” genetics by default (Crews et al. 2014). When paired with additional uncertainties regarding CYP-specific genotype/phenotype associations and the untold numbers of unclassified SNPs, it becomes clear why accurate genetic screening remains challenging, and only one aspect of the therapeutic decision-making process.

Ultimately, the falling cost and broader availability of pyrosequencing technologies support our call for improved RNAseq strategies for personalized medicine, as accurate identification of the patient’s functional genome is a crucial component of precision medicine. While transcriptomic analysis offers superior guidance in the design of personalized therapeutic options, its broad implementation will require technical improvements to sample collection and processing that are also problematic for genomic testing. In this regard, complimentary metabolomics approaches

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 directed at variant-specific metabolism may provide more feasible, short-term improvements to

PGx screening and precision-based approaches to medicine (Beger et al., 2016). Innovative, gene-directed therapeutic technologies such as splice-altering antisense oligonucleotides and

CRISPR/Cas9 genome editing systems may also become feasible tools for manipulating a patient’s transcriptome to optimize therapeutic outcomes. Key examples of splice-switching technology already being investigated to treat human disease are listed in Supplemental Table 2.

In this regard, our group has participated in the development of Eteplirsen, the new antisense Downloaded from oligonucleotide drug which received accelerated approval from the FDA for the treatment of

Duchenne muscular dystrophy (DMD; Syed, 2016; Niks and Aartsma-Rus, 2017). Eteplirsen’s

development evolved from early studies of exon skipping in the murine dystrophin model (Fall et dmd.aspetjournals.org al., 2006; Fletcher et al., 2007; Adams et al., 2007), a canine model of DMD (McClorey et al.,

2006), and in human muscle explants (McClorey et al., 2006). Our group has also employed exon skipping oligomers to refine the immune response-mediated gene expression of IL-10 in an at ASPET Journals on September 30, 2021 Ebola virus lethal challenge mouse model (Mourich and Iversen, 2009; Panchal et al., 2014) and of CTLA-4 in a murine model of autoimmune diabetes (Mourich et al., 2014). We hypothesize that similar splice-altering technology may be useful in re-directing the function of drug metabolizing CYPs, like CYP3A4 (Arora and Iversen, 2001), whose metabolism of drugs like tamoxifen, is linked to genotoxicity (Mahadevan et al., 2006). As our appreciation for transcriptome expansion and the mechanisms of alternative CYP gene splicing evolve, new therapeutic gene editing options are likely to emerge that could scarcely be predicted using genetic testing alone.

In summary, the human cytochrome P450 family transcriptome contains over 965 different variant forms (Table 2), many with common structural features sensitive to alternative splicing events that expand CYP protein diversity. The transcription and processing of CYP gene transcripts is complex and coordinately-regulated within the nucleus by multiple factors, including nuclear receptor (NR) signaling via environmental sensors like the peroxisome proliferator-

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 activated receptors (PPARs), PPARγ and PPARα, which interact with the PGC-1α transcriptional coactivator to regulate oxidative metabolism and mitochondrial biogenesis (Wu et al., 1999,

Monsalve et al., 2000). Multiple steroids, including products of CYP 1A, 1B, 2A, 2B, 2C, 2D, 3A,

7A, 17A, 19A, 24A1 and 51A metabolism, bind NRs leading to interactions with NR coregulators through LxxLL or FxxLF motifs that modulate the assembly of the spliceosome complex and pre- mRNA splicing (Auboeuf et al., 2005). The androgen receptor (AR), which binds multiple metabolites of CYPs 2A, 2C, 2D, 3A, 17A, 19A, and 21A, can also directly interact with nucleolar Downloaded from splicing factors (e.g. U5snRNPs), indicating a receptor-mediated role in transcription that is coupled to pre-mRNA splicing mechanisms (Zhao et al., 2002). Vitamin D receptor (VDR)

activation mediated by metabolites of CYPs 2R, 2J, 3A, 11A, 27A, 24A, 27B can also alter CYP dmd.aspetjournals.org gene expression and splicing, through NR mediated-crosstalk (e.g. PPARs) transduced via interactions with the retinoid X receptor (RXR; Matsuda and Kitagishi, 2013), recruitment of the

NCoA62/SKIP coactivator complex (Zhang et al., 2003) and discrete interactions with the at ASPET Journals on September 30, 2021 heterogeneous nuclear ribonucleoprotein C splicing factor (hnRNPC; Zhou et al., 2014).

Traditional VDR signaling in the nucleus is further refined by several non-traditional NR functions operating near the plasma membrane, which alter gene expression via modulation of key membrane-based paracrine signaling pathways, mediated by agents like Wnt and epidermal growth factor (Larriba et al., 2014). Vitamin A metabolites (or retinoids) of CYPs 1A, 2B, 2C and

26, signaling through the retinoic acid receptor (RAR) and RXR, are also known to guide the recruitment of SC35 coactivators to regulate the alternate splicing of protein kinase delta (PKCδ) among other pre-mRNAs (Apostolatos et al., 2010). Collectively, these data reveal a novel CYP- based mechanism for adaptive transcriptome remodeling, whereby xenobiotics and endogenous substrates, monitored by one of several tissue or disease-specific “CYP clouds”, are metabolized in a coordinated fashion that harmonize NR signaling cascades with alternative gene expression and splicing events that promote adaptive responses to cell stress or stimuli (Figure 4).

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

In conclusion, the human metabolome adapts to substrate burden through the induction of gene transcription, which helps to maintain homeostasis in a well-documented pathway guided by NR binding and signaling events. In this respect, the metabolic response to xenobiotics (via

CYP induction) is adaptive in a manner reminiscent of the immune response to viral antigen; there is a recognition phase of the chemical by the CYP active site, an activation phase when the chemical (or CYP metabolite) interacts with the NR, and an effector phase where the coordinated transcription and splicing of CYP transcripts occurs to feedback-modulate NR signaling. The Downloaded from analogy to the immune response is appropriate here in that specific transcript variants are produced in response to a specific chemical stimulus. The ability to tightly control cellular

homeostasis via NR-mediated gene expression and alternative splicing implies a high order of dmd.aspetjournals.org sophistication operating in what might be considered a primitive, chemical immune response.

Molecules that engage this primitive CYP-based immune system transduce transcriptome-wide biological responses capable of reshaping both the phenotype and “epigenotype” of the cell (via at ASPET Journals on September 30, 2021 the regulation of both coding and noncoding RNA), allowing for reversible, environmental adaptation, as well as imprinting, which in rare cases, may persist trans-generationally (Hochberg et al., 2011). Improved knowledge of both the adaptive and maladaptive epigenome remodeling processes induced by xenobiotics may ultimately help reconcile inter-individual variability in efficacy and toxicity that plague many FDA-approved drugs. These insights will provide new guidance for developing “individualized” therapeutic strategies more sensitive to a patient’s adaptive transcriptome or functional genome, which as exemplified by CYP superfamily of genes, is the ultimate expression of the heritable epigenotype, which appears to remain environmentally- responsive throughout all phases of the human lifecycle.

ACKNOWLEDGMENTS

We would like to thank Dr. Ronald N. Hines (US-EPA) for his thoughtful comments and suggestions concerning this review.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

AUTHORSHIP CONTRIBUTIONS

Participated in research design: Annalora, Iversen and Marcus

Performed data analysis: Annalora and Iversen

Wrote or contributed to the writing of the manuscript: Annalora, Iversen and Marcus

Downloaded from

dmd.aspetjournals.org

at ASPET Journals on September 30, 2021

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

REFERENCES

Abraham JE, Maranian MJ, Driver KE, Platte R, Kalmyrzaev B, Baynes C, Luccarini C,

Shah M, Ingle S, Greenberg D, Early HM, Dunning AM, Pharoah PDP and Caldas C (2010)

CYP2D6 gene variants: association with breast cancer specific survival in a cohort of breast cancer patients from the United kingdom treated with adjuvant tamoxifen. Breast Cancer

Research 12: R64-R75.

Abreu MT, Kantorovich V, Vasiliauskas EA, Gruntmanis U, Matuk R, Daigle K, Chen S, Downloaded from Zehnder D, Lin YC, Yang H, Hewison M, Adams JS. (2004) Measurement of vitamin D levels in inflammatory bowel disease patients reveals a subset of Crohn's disease patients with elevated

1,25-dihydroxyvitamin D and low bone mineral density. Gut 53(8):1129-36. dmd.aspetjournals.org

Adams AM, Harding PL, Iversen PL, Coleman C, Fletcher S, Wilton SD (2007) Antisense oligonucleotide induced exon skipping and the dystrophin gene transcript: cocktails and chemistries. BMC Mol Biol 8: 57-65. at ASPET Journals on September 30, 2021 Allorge D, Chevalier D, Lo-Guidice JM, Cauffiez C, Suard F, Baumann P, Eap CB, Broly

F (2003) Identification of a novel splice-site mutation in the CYP1A2 gene. Br J Clin Pharmacol

56(3): 341-4.

Anandatheerthavarada HK, Addya S, Dwivedi RS, Biswas G, Mullick J, Avadhani NG

(1997) Localization of multiple forms of inducible cytochromes P450 in rat liver mitochondria: immunological characteristics and patterns of xenobiotic substrate metabolism. Arch Biochem

Biophys 339(1): 136-50.

Annalora A, Bobrovnikova-Marjon E, Serda R, Lansing L, Chiu ML, Pastuszyn A, Iyer S,

Marcus CB, Omdahl JL (2004) Rat cytochrome P450C24 (CYP24A1) and the role of F249 in substrate binding and catalytic activity. Arch Biochem Biophys 425(2): 133-46.

Annalora AJ, Goodin DB, Hong WX, Zhang Q, Johnson EF, Stout CD (2010) Crystal structure of CYP24A1, a mitochondrial cytochrome P450 involved in vitamin D metabolism. J Mol

Biol 396(2): 441-51.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

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 splicing of protein kinase CδVIII (PKCδVIII) isoform via splicing factor SC35. J Biol.

Chem 285: 25987-25997.

Arese M, Chen Y, Florkiewicz RZ, Gualandris A, Shen B, Rifkin DB. (1999) Nuclear activities of basic fibroblst growth factor: potentiation of low-serum growth mediated by natural or chimeric nuclear localization signals. Mol Cell Biol 10: 1429-1444. Downloaded from Ariyoshi N, Shimizu Y, Kobayashi Y, Nakamura H, Nakasa H, Nakazawa K, Ishii I, Kitada

M (2007) Identification and partial characterization of a novel CYP2C9 splicing variant encoding

a protein lacking eight amino acid residues. Drug Metab Pharmacokinet 22(3): 187-94. dmd.aspetjournals.org

Arora V, Iversen PL (2001) Redirection of drug metabolism using antisense technology.

Curr Opin Mol Ther 3(3): 249-57. Review.

Auboeuf D, Dowhan DH, Dutertre M, Martin N, Berget SM, O'Malley BW (2005) A subset at ASPET Journals on September 30, 2021 of nuclear receptor coregulators act as coupling proteins during synthesis and maturation of RNA transcripts. Mol Cell Biol 25(13): 5307-16. Review.

Bailey D, Veljkovic K, Yazdanpanah M, Adeli K (2013) Analytical measurement and clinical relevance of vitamin D(3) C3-epimer. Clin Biochem. 46(3): 190-6.

Bajpai P, Srinivasan S, Ghosh J, Nagy L, Wei S, and Guengerich FP (2014) Targeting of splice variants of human cytochrome P450 2C8 to mitochondria and their role in arachidonic acid metabolism and respiratory dysfunction. J Biol Chem 289(43): 29614-30.

Bauer M, Herbarth O, Aust G, and Graebsch C (2005) Molecular cloning and expression of novel alternatively spliced cytochrome P450 2E1 mRNAs in humans. Mol Cell Biochem 280:

201-207.

Bauer M, Herbarth O, Rudzok S, Schmucking E, Muller A, Aust G, and Grabsch C (2007)

Diversity of common alternative splicing variants of human Cytochrome P450 1A1 and their association to carcinogenesis. Int J Oncol 31: 211-218.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Beger RD, Dunn W, Schmidt MA, Gross SS, Kirwan JA, Cascante M, Brennan L, Wishart

DS, Oresic M, Hankemeier T, Broadhurst DI, Lane AN, Suhre K, Kastenmüller G, Sumner SJ,

Thiele I, Fiehn O, Kaddurah-Daouk R; for “Precision Medicine and Pharmacometabolomics Task

Group”-Metabolomics Society Initiative (2016) Metabolomics enables precision medicine: "A

White Paper, Community Perspective". Metabolomics 12(10):149.

Black DL (2000) Protein diversity from alternative splicing: a challenge for bioinformatics and post-genome biology. Cell 103(3): 367-70. Review. Downloaded from Blencowe BJ (2006) Alternative splicing: new insights from global analyses. Cell 126(1):

37-47. Review.

Bouraïma H, Hanoux V, Mittre H, Féral C, Benhaïm A, Leymarie P (2001) Expression of dmd.aspetjournals.org the rabbit cytochrome P450 aromatase encoding gene uses alternative tissue-specific promoters.

Eur J Biochem 268(16): 4506-12.

Brändén G, Sjögren T, Schnecke V, Xue Y (2014) Structure-based ligand design to at ASPET Journals on September 30, 2021 overcome CYP inhibition in drug discovery projects. Drug Discov Today 19(7): 905-11.

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

Carr DF, Alfirevic A, and Pirmoamed M (2014) Pharmacogenomics: Current State-of-the-

Art. Genes 5: 430-443.

Caudle KE, Gammal RS, Whirl-Carrillo M, Hoffman JM, Relling MV, Klein TE (2016)

Evidence and resources to implement pharmacogenetic knowledge for precision medicine. Am J

Health Syst Pharm 73(23):1977-1985.

Chen PH, Lee KW, Hsu CC, Chen JY, Wang YH, Chen KK, Wang HM, Huang HW, Huang

B (2014) Expression of a splice variant of CYP26B1 in betel quid-related oral cancer. Sci World J

2014: 810561.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Chen W, Kubota S, Ujike H, Ishihara T, Seyama Y (1998) A novel Arg362Ser mutation in the sterol 27-hydroxylase gene (CYP27): its effects on pre-mRNA splicing and enzyme activity.

Biochemistry 37(43): 15050-6.

Chinta SJ, Kommaddi RP, Turman CM, Strobel HW, Ravindranath V (2005) Constitutive 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(3): 724-36.

Christmas P, Ursino SR, Fox JW, Soberman RJ (1999) Expression of the CYP4F3 gene. Downloaded from Tissue-specific splicing and alternative promoters generate high and low K(m) forms of leukotriene B(4) omega-hydroxylase. J Biol Chem 274(30): 21191-9.

Christmas P, Jones JP, Patten CJ, Rock D, Zheng Y, Cheng SM, Weber BM, Carlesso N, dmd.aspetjournals.org

Scadden DT, Rettie AE, Soberman RJ (2001) Alternative splicing determines the function of

CYP4F3 by switching substrate specificity. J Biol Chem 276(41): 38166-72.

Costa-Santos M, Kater CE, Dias EP, Auchus RJ (2004) Two intronic mutations cause 17- at ASPET Journals on September 30, 2021 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

Clin Endocrinol Metab 89(1): 43-8.

Cowie P, Hay EA, MacKenzie A (2015) The noncoding human genome and the future of personalised medicine. Expert Rev Mol Med 17: e4.

Cunningham F, Amode MR, Barrell D, Beal K, Billis K, Brent S, Carvalho-Silva D, Clapham

P, Coates G, Fitzgerald S, Gil L, Girón CG, Gordon L, Hourlier T, Hunt SE, Janacek SH, Johnson

N, Juettemann T, Kähäri AK, Keenan S, Martin FJ, Maurel T, McLaren W, Murphy DN, Nag R,

Overduin B, Parker A, Patricio M, Perry E, Pignatelli M, Riat HS, Sheppard D, Taylor K, Thormann

A, Vullo A, Wilder SP, Zadissa A, Aken BL, Birney E, Harrow J, Kinsella R, Muffato M, Ruffier M,

Searle SM, Spudich G, Trevanion SJ, Yates A, Zerbino DR, Flicek P (2015) Ensembl 2015.

Nucleic Acids Res 43: D662-9.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Crews KR, Gaedigk A, Dunnenberger HM, Leeder JS, Klein TE, Caudle KE, Haidar CE,

Shen DD, Callaghan JT, Sadhasivam S, Prows CA, Kharasch ED, Skaar TC (2014) Clinical

Pharmacogenetics Implementation Consortium.. Clinical Pharmacogenetics Implementation

Consortium guidelines for cytochrome P450 2D6 genotype and codeine therapy: 2014 update.

Clin Pharmacol Ther 95(4):376-82. Review.

deMorais SMF, Wilkinson GR, Blaisdell J, Nakamura K, Meyer UA, and Goldstein JA

(1994) The major defect responsible for the polymorphism of S-mephenytoin metabolism in Downloaded from humans. J Biol Chem 269(22): 15419-15422.

Denson J, Wu Y, Yang W and Zhang J (2005) Inter-individual variation of several

cytochrome P450 2D6 splice variants in human liver. Biochem Biophys Res Comm 330: 498-504. dmd.aspetjournals.org

Desrochers M, Christou M, Jefcoate C, Belzil A, Anderson A (1996) New proteins in the rat CYP2B subfamily: presence in liver microsomes of the constitutive CYP2B3 protein and the phenobarbital-inducible protein product of alternatively spliced CYP2B2 mRNA. Biochem at ASPET Journals on September 30, 2021 Pharmacol 52(8): 1311-9.

Diesel B, Seifert M and Radermacher J (2004) Towards a complete picture of splice variants of the gene for 25-hydroxyvitamin D3- 1α-hydroxylase in brain and skin cancer. J Steroid

Biochem Mol Biol 89-90(1-5): 527-532.

Ding S, Lake BG, Friedberg T, Wolf CR (1995) Expression and alternative splicing of the cytochrome P-450 CYP2A7. Biochem J 306 (Pt 1): 161-6.

Doleschall M, Szabó JA, Pázmándi J, Szilágyi Á, Koncz K, Farkas H, Tóth M, Igaz P, Gláz

E, Prohászka Z, Korbonits M, Rácz K, Füst G, Patócs A (2014) Common genetic variants of the human steroid 21-hydroxylase gene (CYP21A2) are related to differences in circulating hormone levels. PLoS One 9(9): e107244.

Dong H, Shertzer HG, Genter MB, Gonzalez FJ, Vasiliou V, Jefcoate C, Nebert DW (2013)

Mitochondrial targeting of mouse NQO1 and CYP1B1 proteins. Biochem Biophys Res Commun

435(4): 727-32.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Elens L, Bouamar R, Hesselink DA, Haufroid V, van der Heiden IP, van Gelder T, van

Schaik RH (2011) A new functional CYP3A4 intron 6 polymorphism significantly affects tacrolimus pharmacokinetics in kidney transplant recipients. Clin Chem 57(11): 1574-83.

Eliasson E, Kenna JG (1996) Cytochrome P450 2E1 is a cell surface autoantigen in halothane hepatitis. Mol Pharmacol 50(3): 573-82.

Elmabsout AA, Kumawat A, Saenz-Méndez P, Krivospitskaya O, Sävenstrand H, Olofsson

PS, Eriksson LA, Strid A, Valen G, Törmä H, Sirsjö A (2012) Cloning and functional studies of a Downloaded from splice variant of CYP26B1 expressed in vascular cells. PLoS One 7(5): e36839.

Faiq MA, Dada R, Sharma R, Saluja D, Dada T (2014) CYP1B1: a unique gene with

unique characteristics. Curr Drug Metab 15(9): 893-914. Review dmd.aspetjournals.org

Fall AM, Johnsen R, Honeyman K, Iversen P, Fletcher S and Wilton SD (2006) Induction of revertant fibres in the mdx mouse using antisense oligonucleotides. Genet Vaccines Ther 4:3-

15. at ASPET Journals on September 30, 2021 Finta C, Zaphiropoulos PG (2000) The human cytochrome P450 3A locus. Gene evolution by capture of downstream exons. Gene 260(1-2): 13-23.

Finta C, Zaphiropoulos PG (2002) Intergenic mRNA molecules resulting from trans- splicing. J Biol Chem 277(8): 5882-90.

Fischer D, Seifert M, Becker S, Ludders D, Cordes T, Reichrath J, Friedrich M (2007) 25-

Hydroxyvitamin D3 1alpha-hydroxylase splice variants in breast cell lines MCF-7 and MCF-10.

Canc Genom Proteom 4(4): 295-300.

Fischer D, Thomé M, Becker S, Cordes T, Diedrich K, Friedrich M, Thill M (2009) 25-

Hydroxyvitamin D3 1alpha-hydroxylase splice variants in benign and malignant ovarian cell lines and tissue. Anticancer Res 29(9): 3627-33.

Fischer D, Becker S, Cordes T, Bücker B, Diedrich K, Friedrich M, Salehin D, Thill M

(2009) Vitamin D-24-hydroxylase in benign and malignant breast tissue and cell lines. Anticancer

Res 29(9): 3641-5.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Flanagan JN, Wang L, Tangpricha V, Reichrath J, Chen TC, Holick MF (2003) Regulation of the 25-hydroxyvitamin D-1alpha-hydroxylase gene and its splice variant. Recent Results

Cancer Res 164: 157-67.

Fletcher S, Honeyman K, Fall AM, Harding PL, Johnson RD, Steinhaus JP, Moulton HM,

Iversen PL, Wilton SD (2007) Morpholino Oligomer-Mediated Exon Skipping Averts the Onset of

Dystrophic Pathology in the mdx Mouse. Mol Ther 15(9): 1587-1592.

García-Roca P, Medeiros M, Reyes H, Rodríguez-Espino BA, Alberú J, Ortiz L, Vásquez- Downloaded from Perdomo M, Elizondo G, Morales-Buenrostro LE, Mancilla Urrea E, Castañeda-Hernández G

(2012) CYP3A5 polymorphism in Mexican renal transplant recipients and its association with

tacrolimus dosing. Arch Med Res 43(4): 283-7. dmd.aspetjournals.org

Garuti R, Lelli N, Barozzini M, Tiozzo R, Dotti MT, Federico A, Ottomano AM, Croce A,

Bertolini S, Calandra S (1996) Cerebrotendinous xanthomatosis caused by two new mutations of the sterol-27-hydroxylase gene that disrupt mRNA splicing. J Lipid Res 37(7): 1459-67. at ASPET Journals on September 30, 2021 Ghosh D, Lo J, Morton D, Valette D, Xi J, Griswold J, Hubbell S, Egbuta C, Jiang W, An

J, Davies HM (2012) Novel aromatase inhibitors by structure-guided design. J Med Chem 55(19):

8464-76.

Gomez A, Nekvindova J, Travica S, Lee MY, Johansson I, Edler D, Mkrtchian S, Ingelman-

Sundberg M (2010) Colorectal cancer-specific cytochrome P450 2W1: intracellular localization, glycosylation, and catalytic activity. Mol Pharmacol 78(6): 1004-11.

Gonzàlez-Porta M, Frankish A, Rung J, Harrow J, Brazma A (2013) Transcriptome analysis of human tissues and cell lines reveals one dominant transcript per gene. Genome Biol

14(7): R70.

Guantes R, Rastrojo A, Neves R, Lima A, Aguado B, Iborra FJ (2015) Global variability in gene expression and alternative splicing is modulated by mitochondrial content. Genome Res

25(5): 633-44.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Guengerich FP (2013) New trends in cytochrome p450 research at the half-century mark.

J Biol Chem 288(24): 17063-4.

Hadfield KD, Newman WG (2012) Pharmacogenetics of aromatase inhibitors.

Pharmacogenomics 13(6): 699-707. Review.

Hampf M, Dao NT, Hoan NT, Bernhardt R (2001) Unequal crossing-over between aldosterone synthase and 11beta-hydroxylase genes causes congenital adrenal hyperplasia. J

Clin Endocrinol Metab 86(9): 4445-52. Downloaded from Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell. 144:

646–674.

Hecker M, Ullrich V (1989) On the mechanism of prostacyclin and thromboxane A2 dmd.aspetjournals.org biosynthesis. J Biol Chem 264(1):141-50.

Hines RN, McCarver DG (2002) The ontogeny of human drug-metabolizing enzymes: phase I oxidative enzymes. J Pharmacol Exp Ther 300(2): 355-60. Review. at ASPET Journals on September 30, 2021 Hochberg Z, Feil R, Constancia M, Fraga M, Junien C, Carel JC, Boileau P, LeBouc Y,

Deal CL, Lillycrop K, Scharfmann R, Sheppard A, Skinner M, Szyf M, Waterland RA, Waxman

DJ, Whitelaw E, Ong K, Albertsson-Wikland K (2011) Child health, developmental plasticity, and epigenetic programming. Endocr Rev 32(2): 159-224. Review.

Horváth HC, Khabir Z, Nittke T, Gruber S, Speer G, Manhardt T, Bonner E, Kallay E (2010)

CYP24A1 splice variants--implications for the antitumorigenic actions of 1,25-(OH)2D3 in colorectal cancer. J Steroid Biochem Mol Biol 121(1-2): 76-9.

Huang Z, Fasco MJ, Figge HL, Keyomarsi K, Kaminsky LS (1996) Expression of cytochromes P450 in human breast tissue and tumors. Drug Metab Dispos 24(8): 899-905.

Huang Z, Fasco MJ, Spivack S and Kaminsky LS (1997) Comparisons of CYP2D messenger RNA splice variant profiles in human lung tumors and normal tissues. Canc Res 57:

2589-2592.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Hwang DY, Hung CC, Riepe FG, Auchus RJ, Kulle AE, Holterhus PM, Chao MC, Kuo MC,

Hwang SJ, Chen HC (2011) CYP17A1 intron mutation causing cryptic splicing in 17α-hydroxylase deficiency. PLoS One 6(9): e25492.

Ibeanu GC, Blaisdell J, Ferguson RJ, Ghanayem BI, Brosen K, Benhamou S, Bouchardy

C, Wilkinson GR, Dayer P and Goldstein JA (1999) A novel transversion in the intron 5 donor splice junction of CYP2C19 and a sequence polymorphism in exon 3 contribute to the poor metabolizer phenotype for the anticonvulsant drug S-mephenytoin. J Pharm Exptl Ther 290(2): Downloaded from 635-640.

Johnson CS, Chung I, Trump DL (2010) Epigenetic silencing of CYP24 in the tumor

microenvironment. J Steroid Biochem Mol Biol 121(1-2): 338-42. dmd.aspetjournals.org

Johnson EF, Stout CD (2013) Structural diversity of eukaryotic membrane cytochrome p450s. J Biol Chem 288(24): 17082-90. Review.

Kimouli M, Gourvas V, Konstantoudaki X, Basta M, Miyakis S, Spandidos DA (2009) The at ASPET Journals on September 30, 2021 effect of an exon 12 polymorphism of the human thromboxane synthase (CYP5A1) gene in stroke patients. Med Sci Monit 15(1): BR30-5.

Knüpfer H, Schmidt R, Stanitz D, Brauckhoff M, Schönfelder M, Preiss R (2004) CYP2C and IL-6 expression in breast cancer. Breast 13(1): 28-34.

Kommaddi RP, Turman CM, Moorthy B, Wang L, Strobel HW, Ravindranath V (2007) An alternatively spliced cytochrome P4501A1 in human brain fails to bioactivate polycyclic aromatic hydrocarbons to DNA-reactive metabolites. J Neurochem 102(3): 867-77.

Kramlinger VM, Nagy LD, Fujiwara R, Johnson KM, Phan TT, Xiao Y, Enright JM, Toomey

MB, Corbo JC, Guengerich FP (2016) Human cytochrome P450 27C1 catalyzes 3,4-desaturation of retinoids. FEBS Lett. 590(9): 1304-12.

Kristensen VN, Harada N, Yoshimura N, Haraldsen E, Lonning PE, Erikstein B, Kåresen

R, Kristensen T, Børresen-Dale AL (2000) Genetic variants of CYP19 (aromatase) and breast cancer risk. Oncogene 19(10): 1329-33.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Kuehl P, Zhang J, Lin Y, Lamba J, Assem M, Schuetz J, Watkins PB, Daly A, Wrighton

SA, Hall SD, Maurel P, Relling M, Brimer C, Yasuda K, Venkataramanan R, Strom S, Thummel

K, Boguski MS, Schuetz E (2001) Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression. Nat Genet 27(4): 383-91.

La Cognata V, Iemmolo R, D’Agata V, Scuderi S, Drago F, Zappia M, and Cavallaro S

(2014) Increasing the coding potential of genomes through alternative splicing: The case of

PARK2 gene. Current Genomics 15: 203-216. Downloaded from Lamb DC and Waterman MR (2013) Unusual properties of the cytochrome P450 superfamily. Philos Trans R Soc Lond B Biol Sci 368(1612): 20120434.

Lander ES, Linton LM et al (2001) Initial sequencing and analysis of the human genome. Nature dmd.aspetjournals.org

409: 860-921.

Larriba MJ, González-Sancho JM, Bonilla F, Muñoz A (2014) Interaction of vitamin D with membrane-based signaling pathways. Front Physiol 5: 60. Review. at ASPET Journals on September 30, 2021 Lee HH (2013) Variants of the CYP21A2 and CYP21A1P genes in congenital adrenal hyperplasia. Clin Chim Acta 418: 37-44.

Lee SJ, van der Heiden IP, Goldstein JA, van Schaik RH (2007) A new CYP3A5 variant,

CYP3A5*11, is shown to be defective in nifedipine metabolism in a recombinant cDNA expression system. Drug Metab Dispos 35(1): 67-71.

Lejeune F, Maquat LE (2005) Mechanistic links between nonsense-mediated mRNA decay and pre-mRNA splicing in mammalian cells. Curr Opin Cell Biol 17(3): 309-15. Review.

Leung YK, Lau KM, Modley J, JiangZ, and Ho SM (2005) Overexpression of Cytochrome

P450 1A1 and its novel spliced variant in ovarian cancer cells: Alternative subcellular enzyme compartmentalization may contribute to carcinogenesis. Cancer Res 65: 3726-3734.

Lewiñska M, Zmrzljak UP, Rozman D (2013) Low nucleotide variability of CYP51A1 in humans: meta-analysis of cholesterol and bile acid synthesis and xenobiotic metabolism pathways. Acta Chim Slov 60(4): 875-83.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Li F, Jiang C, Larsen MC, Bushkofsky J, Krausz KW, Wang T, Jefcoate CR, Gonzalez FJ

(2014) Lipidomics reveals a link between CYP1B1 and SCD1 in promoting obesity. J Proteome

Res 13(5): 2679-87.

Li L, Yin Z, Liu J, Li G, Wang Y, Yan J, Zhou H (2013) CYP46A1 T/C polymorphism associated with the APOE ε4 allele increases the risk of Alzheimer's disease. J Neurol 260(7):

1701-8.

Li M, Wang W, Li Y, Wang L, Shen X, Tang Z (2013) CYP46A1 intron-2T/C polymorphism Downloaded from and Alzheimer's disease: an updated meta-analysis of 16 studies including 3,960 cases and 3,828 controls. Neurosci Lett 549: 18-23.

Lin L, Ercan O, Raza J, Burren CP, Creighton SM, Auchus RJ, Dattani MT, Achermann dmd.aspetjournals.org

JC (2007) Variable phenotypes associated with aromatase (CYP19) insufficiency in humans. J

Clin Endocrinol Metab 92(3): 982-90.

Lin YS, Dowling ALS, Quigley SD, Farin FM, Zhang J, Lamba J, Schuetz EG and Thummel at ASPET Journals on September 30, 2021 KE (2002) Co-Regulation of CYP3A4 and CYP3A5 and contribution to hepatic and intestinal midazolam metabolism. Mol Pharm 62: 162-172.

Liu L, Bai YX, Zhou JH, Sun XW, Sui H, Zhang WJ, Yuan HH, Xie R, Wei XL, Zhang TT,

Huang P, Li YJ, Wang JX, Zhao S, Zhang QY (2013) A polymorphism at the 3'-UTR region of the aromatase gene is associated with the efficacy of the aromatase inhibitor, anastrozole, in metastatic breast carcinoma. Int J Mol Sci 14(9): 18973-88.

Liu X, Low SK, Boddy AV (2016) The implications of genetic variation for the pharmacokinetics and pharmacodynamics of aromatase inhibitors. Expert Opin Drug Metab

Toxicol 12(8):851-63.

Liu YL, Xu Y, Li F, Chen H, Guo SL (2013) CYP2A6 deletion polymorphism is associated with decreased susceptibility of lung cancer in Asian smokers: a meta-analysis. Tumour Biol

34(5): 2651-7.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Lu Y, Mo C, Zeng Z, Chen S, Xie Y, Peng Q, He Y, Deng Y, Wang J, Xie L, Zeng J, Li S,

Qin X (2013) CYP2D6*4 allele polymorphism increases the risk of Parkinson's disease: evidence from meta-analysis. PLoS One 8(12): e84413.

Lum DW, Perel P, Hingorani AD, Holmes MV (2013) CYP2D6 genotype and tamoxifen response for breast cancer: a systematic review and meta-analysis. PLoS One 8(10):e76648.

Maas RM, Reus K, Diesel B, Steudel WI, Feiden W, Fischer U, Meese E (2001)

Amplification and expression of splice variants of the gene encoding the P450 cytochrome 25- Downloaded from hydroxyvitamin D(3) 1,alpha-hydroxylase (CYP 27B1) in human malignant glioma. Clin Cancer

Res. 7(4): 868-75.

Mahadevan B, Arora V, Schild LJ, Keshava C, Cate ML, Iversen PL, Poirier MC, Weston dmd.aspetjournals.org

A, Pereira C, Baird WM (2006) Reduction in tamoxifen-induced CYP3A2 expression and DNA adducts using antisense technology. Mol Carcinog 45(2): 118-25.

Mangin M, Sinha R, Fincher K. (2014) Inflammation and vitamin D: the infection at ASPET Journals on September 30, 2021 connection. Inflamm Res 63(10):803-19. Review.

Matsuda S, Kitagishi Y (2013) Peroxisome proliferator-activated receptor and vitamind receptor signaling pathways in cancer cells. Cancers (Basel) 5(4): 1261-70.

McGlincy NJ, Smith CW (2008) Alternative splicing resulting in nonsense-mediated mRNA decay: what is the meaning of nonsense? Trends Biochem Sci 33(8): 385-93. Review.

McClorey G, Moulton HM, Iversen PL, Fletcher S. and Wilton SD (2006) Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD. Gene Ther 13(19): 1373-1381.

McClorey G, Fall AM, Moulton HM, Iversen PL, Rasko JE, Ryan M, Fletcher S and Wilton

SD (2006) Induced dystrophin exon skipping in human muscle explants. Neuromuscul Disord (9-

10): 583-590.

Meyer UA (2007) Endo-xenobiotic crosstalk and the regulation of cytochromes P450. Drug

Metab Rev 39(2-3): 639-46. Review.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Miles JS, McLaren AW, Wolf CR (1989) Alternative splicing in the human cytochrome

P450IIB6 gene generates a high level of aberrant messages. Nucleic Acids Res 17(20): 8241-55.

Mitrpant C, Fletcher S, Iversen P and Wilton SD (2009) By-passing the nonsense mutation in the 4(CV) mouse model of muscular dystrophy by inducing exon skipping. J Gene Med 11: 46-

56.

Monsalve M, Wu Z, Adelmant G, Puigserver P, Fan M and Spiegelman BM (2000) Direct coupling of transcription and mRNA processing through the thermogenic coactivator PGC-1. Mol. Downloaded from Cell 6(2): 307-316.

Mort M, Ivanov D, Cooper DN, Chuzhanova NA (2008) A meta-analysis of nonsense

mutations causing human genetic disease. Hum Mutation 29: 1037-1047. dmd.aspetjournals.org

Mourich DV and Iversen, PL (2009) Splicing in the immune system: potential targets for therapeutic intervention by antisense mediated alternative splicing. Current Opinion in Molecular

Therapeutics 11(2): 124-132. at ASPET Journals on September 30, 2021 Mourich DV, Oda SK, Schnell FJ, Crumley SL, Hauck LL, Moentenich CA, Marshall NB,

Hinrichs DJ, and Iversen PL (2014) Alternate Splice Forms of CTLA-4 Induced by Antisense

Mediated Splice-Switching Influences Autoimmune Diabetes Susceptibility in NOD Mice. Nucleic

Acid Ther 24(2): 114-26.

Muindi JR, Nganga A, Engler KL, Coignet LJ, Johnson CS, Trump DL (2007) CYP24 splicing variants are associated with different patterns of constitutive and calcitriol-inducible

CYP24 activity in human prostate cancer cell lines. J. Steroid Biochem Mol Biol 103(3-5): 334-7.

Nakayama T, Soma M, Saito S, Honye J, Yajima J, Rahmutula D, Kaneko Y, Sato M,

Uwabo J, Aoi N, Kosuge K, Kunimoto M, Kanmatsuse K, Kokubun S (2002) Association of a novel single nucleotide polymorphism of the prostacyclin synthase gene with myocardial infarction. Am

Heart J 143(5): 797-801.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Nakayama T, Soma M, Watanabe Y, Hasimu B, Sato M, Aoi N, Kosuge K, Kanmatsuse

K, Kokubun S, Marrow JD, Oates JA (2002) Splicing mutation of the prostacyclin synthase gene in a family associated with hypertension. Biochem Biophys Res Commun. 297(5): 1135-9.

Nelson DR (2003) Comparison of P450s from human and fugu: 420 million years of vertebrate P450 evolution. Arch Biochem Biophys 409(1): 18-24.

Nelson DR, Zeldin DC, Hoffman SM, Maltais LJ, Wain HM, Nebert DW (2004) Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature Downloaded from recommendations for genes, pseudogenes and alternative-splice variants. Pharmacogenetics

14(1): 1-18. Review.

Nhamburo PT, Kimura S, McBride OW, Kozak CA, Gelboin HV, Gonzalez FJ (1990) The dmd.aspetjournals.org human CYP2F gene subfamily: identification of a cDNA encoding a new cytochrome P450, cDNA- directed expression, and chromosome mapping. Biochemistry 29(23): 5491-9.

Niks EH, Aartsma-Rus A (2017) Exon skipping: a first in class strategy for Duchenne at ASPET Journals on September 30, 2021 muscular dystrophy. Expert Opin Biol Ther. 17(2):225-236.

Ohgiya S, Komori M, Ohi H, Shiramatsu K, Shinriki N, Kamataki T (1992) Six-base deletion occurring in messages of human cytochrome P-450 in the CYP2C subfamily results in reduction of tolbutamide hydroxylase activity. Biochem Int 27(6): 1073-81.

Okino ST, Quattrochi LC, Pendurthi UR, McBride OW, Tukey RH (1987) Characterization of multiple human cytochrome P-450 1 cDNAs. The chromosomal localization of the gene and evidence for alternate RNA splicing. J Biol Chem 262(33): 16072-9. Erratum in: J Biol Chem

(1988) 263(5): 2576.

Pagliarini V, Naro C, Sette C (2015) Splicing Regulation: A Molecular Device to Enhance

Cancer Cell Adaptation. Biomed Res Int 2015: 543067. Review.

Panchal RG, Ulrich R, Bradfute SB, Lane D, Ruthel G, Kenny TA, Iversen PL, Anderson

AO, Gussio R, Raschke WC and Bavari S (2009) Reduced Expression of CD45 Protein Tyrosine

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Phosphatase Provides Protection Against Anthrax Pathogenesis. J Biol Chem 284(19): 12874-

12885.

Panchal RG, Mourich DV, Bradfute S, Hauck LL, Warfield KL, Iversen PL, and Bavari S

(2014) Induced IL-10 Splice Altering Approach to Antiviral Drug Discovery. Nucleic Acid

Therapeutics 24(3): 179-185.

Park JW, Reed JR, Backes WL (2015) The Localization of Cytochrome P450s CYP1A1 and CYP1A2 into Different Lipid Microdomains Is Governed by Their N-terminal and Internal Downloaded from Protein Regions. J Biol Chem 290(49): 29449-60.

Peng X, Tiwari N, Roy S, Yuan L, Murillo G, Mehta RR, Benya RV, Mehta RG (2012)

Regulation of CYP24 splicing by 1,25-dihydroxyvitamin D₃ in human colon cancer cells. J dmd.aspetjournals.org

Endocrinol 212(2): 207-15.

Pepe CM, Saraco NI, Baquedano MS, Guercio G, Vaiani E, Marino R, Pandey AV, Flück

CE, Rivarola MA, Belgorosky A (2007) The cytochrome P450 aromatase lacking exon 5 is at ASPET Journals on September 30, 2021 associated with a phenotype of nonclassic aromatase deficiency and is also present in normal human steroidogenic tissues. Clin Endocrinol (Oxf) 67(5): 698-705.

Pikuleva IA, Waterman MR (2013) Cytochromes p450: roles in diseases. J Biol Chem

288(24): 17091-8. Review.

Poliseno L, Salmena L, Zhang J, Carver B, Haveman WJ, Pandolfi PP (2010) A coding independent function of gene and pseudogene mRNAs regulates tumour biology. Nature 465

(7301): 1033–1038.

Qiao J, Han B, Liu BL, Liu W, Wu JJ, Pan CM, Jiang H, Gu T, Jiang BR, Zhu H, Lu YL,

Wu WL, Chen MD, Song HD. (2011) A unique exonic splicing mutation in the CYP17A1 gene as the cause for steroid 17{alpha}-hydroxylase deficiency. Eur J Endocrinol 164(4):627-33.

Qu G, Dong X, Piazza CL, Chalamcharla VR, Lutz S, Curcio MJ, Belfort M. (2014) RNA-

RNA interactions and pre-mRNA mislocalization as drivers of group II intron loss from nuclear genomes. Proc Natl Acad Sci USA 111(18):6612-7.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Raj A, van Oudenaarden A. (2008) Nature, nurture, or chance: stochastic gene expression and its consequences. Cell 135(2):216-26. Review.

Rappaport N, Twik M, Nativ N, Stelzer G, Bahir I, Stein TI, Safran M, Lancet D. (2014)

MalaCards: A Comprehensive Automatically-Mined Database of Human Diseases. Curr Protoc

Bioinformatics 47:1.24.1-1.24.19.

Rat E, Billaut-Laden I, Allorge D, Lo-Guidice JM, Tellier M, Cauffiez C, Jonckheere N, van

Seuningen I, Lhermitte M, Romano A, Guéant JL, Broly F. (2006) Evidence for a functional genetic Downloaded from polymorphism of the human retinoic acid-metabolizing enzyme CYP26A1, an enzyme that may be involved in spina bifida. Birth Defects Res A Clin Mol Teratol 76(6):491-8.

Ren S, Nguyen L, Wu S, Encinas C, Adams JS, and Hewison M. (2005) Alternative dmd.aspetjournals.org splicing of vitamin D-24-hydroxylase: a novel mechanism for the regulation of extrarenal 1,25- dihydroxyvitamin D synthesis. J. Biol. Chem 280 (21), 20604-20611.

Rhieu SY, Annalora AJ, Wang G, Flarakos CC, Gathungu RM, Vouros P, Sigüeiro R, at ASPET Journals on September 30, 2021 Mouriño A, Schuster I, Palmore GT, Reddy GS. (2013) Metabolic stability of 3-epi-1α,25- dihydroxyvitamin D3 over 1 α 25-dihydroxyvitamin D3: metabolism and molecular docking studies using rat CYP24A1. J Cell Biochem 114(10):2293-305.

Robins T, Carlsson J, Sunnerhagen M, Wedell A, Persson B. (2006) Molecular model of human CYP21 based on mammalian CYP2C5: structural features correlate with clinical severity of mutations causing congenital adrenal hyperplasia. Mol Endocrinol 20(11):2946-64.

Rogozin IB, Carmel L, Csuros M, Koonin EV. (2012) Origin and evolution of spliceosomal introns. Biol Direct 7:11. Review.

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.

Salzman J, Gawad C, Wang PL, Lacayo N, Brown PO. (2012) Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. PLoS One

7(2):e30733.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Sangar MC, Anandatheerthavarada HK, Martin MV, Guengerich FP and Avadhani NG.

(2010) Identification of genetic variants of human cytochrome P450 2D6 with impaired mitochondrial targeting. Mol Genet Metab 99(1):90-97.

Santosh B, Varshney A, Yadava PK. (2015) Non-coding RNAs: biological functions and applications. Cell Biochem Funct 33(1):14-22.

Sarasquete ME, García-Sanz R, Marín L, Alcoceba M, Chillón MC, Balanzategui A,

Santamaria C, Rosiñol L, de la Rubia J, Hernandez MT, Garcia-Navarro I, Lahuerta JJ, González Downloaded from M, San Miguel JF. (2008) Bisphosphonate-related osteonecrosis of the jaw is associated with polymorphisms of the cytochrome P450 CYP2C8 in multiple myeloma: a genome-wide single

nucleotide polymorphism analysis. Blood 112(7):2709-12. dmd.aspetjournals.org

Satyanarayana CR, Devendran A, Jayaraman M, Mannu J, Mathur PP, Gopal SD,

Rajagopal K, Chandrasekaran A. (2009) Influence of the genetic polymorphisms in the 5' flanking and exonic regions of CYP2C19 on proguanil oxidation. Drug Metab Pharmacokinet 24(6):537- at ASPET Journals on September 30, 2021 48.

Scheible C, Thill M, Baum S, Solomayer E, Friedrich M. (2014) Implication of CYP24A1 splicing in breast cancer. Anticancer Agents Med Chem 14(1):109-14.

Searles Nielsen S, Bammler TK, Gallagher LG, Farin FM, Longstreth W Jr, Franklin GM,

Swanson PD, Checkoway H. (2013) Genotype and age at Parkinson disease diagnosis. Int J Mol

Epidemiol Genet 4(1):61-9.

Seifert M, Tilgen W, Reichrath J. (2009) Expression of 25-hydroxyvitamin D-1alpha- hydroxylase (1alphaOHase, CYP27B1) splice variants in HaCaT keratinocytes and other skin cells: modulation by culture conditions and UV-B treatment in vitro. Anticancer Res 29(9):3659-

67.

Sevrioukova IF, Poulos TL. (2013) Dissecting cytochrome P450 3A4-ligand interactions using ritonavir analogues. Biochemistry 52(26):4474-81.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Sharaf S, Hafez M, ElAbd D, Ismail A, Thabet G, Elsharkawy M. (2014) High frequency of splice site mutation in 21-hydroxylase deficiency children. J Endocrinol Invest 38(5):505-11.

Sheikh SA, Waryah AM, Narsani AK, Shaikh H, Gilal IA, Shah K, Qasim M, Memon AI,

Kewalramani P, Shaikh N. (2014) Mutational spectrum of the CYP1B1 gene in Pakistani patients with primary congenital glaucoma: novel variants and genotype-phenotype correlations. Mol Vis

20:991-1001.

Shuldiner AR, O'Connell JR, Bliden KP, Gandhi A, Ryan K, Horenstein RB, Damcott CM, Downloaded from Pakyz R, Tantry US, Gibson Q, Pollin TI, Post W, Parsa A, Mitchell BD, Faraday N, Herzog W,

Gurbel PA. (2009) Association of cytochrome P450 2C19 genotype with the antiplatelet effect and

clinical efficacy of clopidogrel therapy. JAMA 302(8):849-57. dmd.aspetjournals.org

Slavotinek AM, Mehrotra P, Nazarenko I, Tang PL, Lao R, Cameron D, Li B, Chu C, Chou

C, Marqueling AL, Yahyavi M, Cordoro K, Frieden I, Glaser T, Prescott T, Morren MA, Devriendt

K, Kwok PY, Petkovich M, Desnick RJ. (2013) Focal facial dermal dysplasia, type IV, is caused at ASPET Journals on September 30, 2021 by mutations in CYP26C1. Hum Mol Genet 22(4):696-703.

Solomon O, Oren S, Safran M, Deshet-Unger N, Akiva P, Jacob-Hirsch J, Cesarkas K,

Kabesa R, Amariglio N, Unger R, Rechavi G, Eyal E. (2013) Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR). RNA 19(5):591-604.

Stelzer G, Dalah I, Iny Stein T, Satanower Y, Rosen N, Nativ N, Oz-Levi D, Olender T,

Belinky F, Bahir I, Krug H, Perco P, Mayer B, Kolker E, Safran M and Lancet, D. (2011) In-silico

Human Genomics with GeneCards. Human Genomics 5(6):709-17.

St. Laurent G, Vyatkiin Y, and Kapranov P. (2014) Dark Matter RNA illuminates the puzzle of genome-wide association studies. BMC Medicine 12:97-105.

Stenstedt K, Hallstrom M, Johansson I, Ingelman-Sundberg M, Ragnhammar P, Edler D.

(2012) The expression of CYP2W1: a prognostic marker in colon cancer. Anticancer Res

32(9):3869-74.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Stoilov I, Akarsu AN, Alozie I, Child A, Barsoum-Homsy M, Turacli ME, Or M, Lewis RA,

Ozdemir N, Brice G, Aktan SG, Chevrette L, Coca-Prados M, Sarfarazi M. (1998) Sequence analysis and homology modeling suggest that primary congenital glaucoma on 2p21 results from mutations disrupting either the hinge region or the conserved core structures of cytochrome

P4501B1. Am J Hum Genet 62(3):573-84.

Sun W, Li Y, Li J, Zhang Z, Zhu W, Liu W, Cai Q, Wang X, Cao L, Bai W, Fan X, Ma M,

Guo R, Liu X, Xu G. (2014) Variant recurrent risk among stroke patients with different CYP2C19 Downloaded from phenotypes and treated with clopidogrel. Platelets 26(6):558-62.

Syed YY (2016) Eteplirsen: First Global Approval. Drugs 76(17):1699-1704.

Szabó JA, Szilágyi Á, Doleschall Z, Patócs A, Farkas H, Prohászka Z, Rácz K, Füst G, dmd.aspetjournals.org

Doleschall M. (2013) Both positive and negative selection pressures contribute to the polymorphism pattern of the duplicated human CYP21A2 gene. PLoS One 8(11):e81977.

Tanwar M, Dada T, Sihota R, Das TK, Yadav U, Dada R. (2009) Mutation spectrum of at ASPET Journals on September 30, 2021 CYP1B1 in North Indian congenital glaucoma patients. Mol Vis 15:1200-9.

Thierry-Mieg D, Thierry-Mieg J. (2006) AceView: a comprehensive cDNA-supported gene and transcripts annotation. Genome Biol 1:S12.1-14.

Tian D, Zhang ZQ. (2011) 2 Novel deletions of the sterol 27-hydroxylase gene in a Chinese

Family with Cerebrotendinous Xanthomatosis. BMC Neurol 11:130.

Toscano C, Klein K, Blievernicht J, Schaeffeler E, Saussele T, Raimundo S, Eichelbaum

M, Schwab M, Zanger UM. (2006) Impaired expression of CYP2D6 in intermediate metabolizers carrying the *41 allele caused by the intronic SNP 2988G>A: evidence for modulation of splicing events. Pharmacogenet Genomics 16(10):755-66.

Tournel G, Cauffiez C, Billaut-Laden I, Allorge D, Chevalier D, Bonnifet F, Mensier E,

Lafitte JJ, Lhermitte M, Broly F, Lo-Guidice JM (2007) Molecular analysis of the CYP2F1 gene: identification of a frequent non-functional allelic variant. Mutat Res 617(1-2): 79-89.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Tralau T, Luch A (2013) The evolution of our understanding of endo-xenobiotic crosstalk and cytochrome P450 regulation and the therapeutic implications. Expert Opin Drug Metab

Toxicol 9(12): 1541-54. Review.

Trump DL, Muindi J, Fakih M, Yu WD, Johnson CS (2006) Vitamin D compounds: clinical development as cancer therapy and prevention agents. Anticancer Res 26(4A): 2551-6.

Turman CM, Hatley JM, Ryder DJ, Ravindranath V, Strobel HW (2006) Alternative splicing within the human cytochrome P450 superfamily with an emphasis on the brain: The convolution Downloaded from continues. Expert Opin Drug Metab Toxicol 2(3): 399-418. Review.

Vanselow J, Zsolnai A, Fésüs L, Fürbass R, Schwerin M (1999) Placenta-specific

transcripts of the aromatase encoding gene include different untranslated first exons in sheep and dmd.aspetjournals.org cattle. Eur J Biochem 265(1): 318-24.

Verrips A, Steenbergen-Spanjers GC, Luyten JA, Wevers RA, Wokke JH, Gabreëls FJ,

Wolthers BG, van den Heuvel LP (1997) Exon skipping in the sterol 27-hydroxylase gene leads at ASPET Journals on September 30, 2021 to cerebrotendinous xanthomatosis. Hum Genet 100(2): 284-6.

Walsh AA, Szklarz GD, Scott EE (2013) Human cytochrome P450 1A1 structure and utility in understanding drug and xenobiotic metabolism. J Biol Chem 288(18): 12932-43.

Wang A, Stout CD, Zhang Q, Johnson EF (2015) Contributions of Ionic Interactions and

Protein Dynamics to Cytochrome P450 2D6 (CYP2D6) Substrate and Inhibitor Binding. J Biol

Chem 290(8): 5092-104.

Wang D, Poi MJ, Sun X, Gaedigk A, Leeder JS, Sadee W (2014) Common CYP2D6 polymorphisms affecting alternative splicing and transcription: long-range haplotypes with two regulatory variants modulate CYP2D6 activity. Hum Mol Genet 23(1): 268-78.

Wang LH, Tazawa R, Lang AQ, Wu KK (1994) Alternate splicing of human thromboxane synthase mRNA. Arch Biochem Biophys 315(2): 273-8.

Wang W, Fu JF, Gong FQ, Zhu WH, Shen Z (2011) Rare hypertension as a result of

17alpha-hydroxylase deficiency. J Pediatr Endocrinol Metab 24(5-6): 333-7. Review.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Wang Z and Burge CB (2008) Splicing regulation: From a parts list of regulatory elements to an integrated splicing code. RNA 14: 802-813.

Warner SC, Finta C, Zaphiropoulos PG (2001) Intergenic transcripts containing a novel human cytochrome P450 2C exon 1 spliced to sequences from the CYP2C9 gene. Mol Biol Evol

18(10): 1841-8.

Wiemann S, Kolb-Kokocinski A, Poustka A (2005) Alternative pre-mRNA processing regulates cell-type specific expression of the IL4l1 and NUP62 genes. BMC Biol 3:16. Review. Downloaded from Wu AH (2011) Drug metabolizing enzymatic activities versus genetic variances for drug of clinical pharmacogenomics relevance. Clinical Proteomics 8: 12-21.

Wu ZL, Bartleson CJ, Ham AJ, Guengerich FP (2006) Heterologous expression, dmd.aspetjournals.org purification, and properties of human cytochrome P450 27C1. Arch Biochem Biophys 445(1): 138-

46.

Wu S, Ren S, Nguyen L, Adams JS, Hewison M (2007) Splice variants of the CYP27b1 at ASPET Journals on September 30, 2021 gene and the regulation of 1,25-dihydroxyvitamin D3 production. Endocrinology 148(7): 3410-8.

Wu Z, Puigserver P, Andersson U, Zhang C, Adelmant G, Mootha V, Troy A, Cinti S,

Lowell B, Scarpulla RC, Spiegelman BM (1999) Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 98(1): 115-24.

Yamaguchi H, Nakazato M, Miyazato M, Toshimori H, Oki S, Shimizu K, Suiko M,

Kangawa K, Matsukura S (1998) Identification of a novel splicing mutation and 1-bp deletion in the 17alpha-hydroxylase gene of Japanese patients with 17alpha-hydroxylase deficiency. Hum

Genet 102(6): 635-9.

Yamano S, Nhamburo PT, Aoyama T, Meyer UA, Inaba T, Kalow W, Gelboin HV, McBride

OW, Gonzalez FJ (1989) cDNA cloning and sequence and cDNA-directed expression of human

P450 IIB1: identification of a normal and two variant cDNAs derived from the CYP2B locus on chromosome 19 and differential expression of the IIB mRNAs in human liver. Biochemistry 28(18):

7340-8.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Zanger UM, Schwab M (2013) Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol Ther 138(1):

103-41. Review.

Zaphiropoulos PG (1997) Exon skipping and circular RNA formation in transcripts of the human cytochrome P-450 2C18 gene in epidermis and of the rat androgen binding protein gene in testis. Mol Cell Biol 17(6): 2985-93.

Zaphiropoulos PG (1999) RNA molecules containing exons originating from different Downloaded from members of the cytochrome P450 2C gene subfamily (CYP2C) in human epidermis and liver.

Nucleic Acids Res 27(13): 2585-90.

Zhang C, Dowd DR, Staal A, Gu C, Lian JB, van Wijnen AJ, Stein GS, MacDonald PN dmd.aspetjournals.org

(2003) Nuclear coactivator-62 kDa/Ski-interacting protein is a nuclear matrix-associated coactivator that may couple vitamin D receptor-mediated transcription and RNA splicing. J Biol

Chem 278(37): 35325-36 at ASPET Journals on September 30, 2021 Zhang C, Wang L, Liao Q, Zhang L, Xu L, Chen C, Ye H, Xu X, Ye M, Duan S (2013)

Genetic associations with hypertension: meta-analyses of six candidate genetic variants. Genet

Test Mol Biomarkers 17(10): 736-42.

Zhao Y, Goto K, Saitoh M, Yanase T, Nomura M, Okabe T, Takayanagi R and Nawata H

(2002) Activation function-1 domain of androgen receptor contributes to the interaction between subnuclear splicing factor compartment and nuclear receptor compartment: identification of the p102 U5 small nuclear ribonucleoprotein particle-binding protein as a coactivator for the receptor.

J Biol Chem 277: 30031-30039.

Zhao B and Waterman MR (2011) Moonlighting cytochrome P450 monooxygenases.

IUBMB Life 63(7): 473-7.

Zhou C, Zhou D, Esteban J, Murai J, Siiteri PK, Wilczynski S, Chen S (1996) Aromatase gene expression and its exon I usage in human breast tumors. Detection of aromatase messenger

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

RNA by reverse transcription-polymerase chain reaction. J Steroid Biochem Mol Biol 59(2): 163-

71.

Zhou R, Chun RF, Lisse TS, Garcia AJ, Xu J, Adams JS, Hewison M. (2014) Vitamin D and alternative splicing of RNA. J Steroid Biochem Mol Biol S0960-0760(14): 00224-6.

Zhou SF, Liu JP, Chowbay B (2009) Polymorphism of human cytochrome P450 enzymes and its clinical impact. Drug Metab Rev 41(2): 89-295. Review.

Zhou SF (2009) Polymorphism of human cytochrome P450 2D6 and its clinical Downloaded from significance: Part I. Clin Pharmacokinet 48(11):689-723. Review.

Zimmerly S, Semper C (2015) Evolution of group II introns. Mob DNA 6: 7.

dmd.aspetjournals.org

at ASPET Journals on September 30, 2021

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

FOOTNOTES

1.) This research was supported by start-up funds from the Office of Research and the College of

Agricultural Sciences at Oregon State University (Corvallis, OR) awarded to Drs. Marcus and

Iversen.

Downloaded from

dmd.aspetjournals.org

at ASPET Journals on September 30, 2021

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

FIGURE LEGENDS

Figure 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 CYP transcript variants and B.) SNPs for each of the 57 human

Cytochrome P450 genes are shown, based on a meta-analysis of information of the NCBI’s

Ensembl, Pubmed and Aceview databases and the GeneCards.org website. Total transcript variants for individual CYP genes identified in this study are shown superimposed (in pink) over Downloaded from the current number listed in the Aceview database alone (in purple). The expansion in variant numbers we identified highlights the challenge of predicting inter-individual variability in

polymorphic CYP gene splicing, based on existing transcript databases. Radar plot comparison dmd.aspetjournals.org of the total human SNPs associated (as reported by Genecards.org), revealed CYP5A1 (or thromboxane synthase 1 (TBXAS1) as the most polymorphic CYP gene (5274), with CYPs 2C19

(2467), 7B1 (3400), 19A1 (2178) and 39A1 (2129) showing higher rates of polymorphism than at ASPET Journals on September 30, 2021 the average CYP gene (~850 mutations per gene). C.) A bar graph representation of alternative

CYP 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 CYP genes. CYPs 21A2 (0.33), 27B1 (0.15) and CYP2D6 (0.11) also skew above normal for this ratio (~0.05 alt-transcripts per SNP). Improved understanding of the

SNP-based mechanisms that alter CYP gene splicing will facilitate the identification of novel SNP biomarkers that are predictive of drug interactions and human disease.

Figure 2. Tissue-specific and Transformation-sensitive Alternative Splicing of CYP1A1 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 exon 6 (Kommadi et al., 2007).

This shortened form of the enzyme is spectrally-active, but does not metabolize benzo(a)pyrene to toxic metabolites in brain. Structural analysis of the CYP1A1 crystal structure (PDB: 4I8V)

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 indicates removal of exon 6 (in yellow ribbon) would: 1) expand the opening of the pw2b substrate access channel; 2) reshape the ligand binding pocket by altering the β1-4 sheet; and 3) alter the redox partner binding surface via elimination of the K’ helix. A similar pattern of tissue-specific, alternative exon 2 usage in CYP1A1 has been 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-unique splice variant with diminished estradiol metabolism that localizes to the nucleus and mitochondria, rather than the ER. Cryptic intron removal in exon 2 eliminates Downloaded from 28 amino acid residues among helices E, F, and the E-F loop (shown in transparent gold ribbon), which putatively expands the opening of the pw3 substrate access channels located above the

heme center, and helix I. dmd.aspetjournals.org

Figure 3. Alternative-Splicing of the Human CYP24A1 Gene: Tissue- and Tumor-Specific

Exon 1, 2 and 10 Inclusion – CYP24A1 is a mitochondrial P450 comprised of 11 coding exons at ASPET Journals on September 30, 2021 that metabolizes the vitamin D hormone to regulate its role in calcium homeostasis, cell growth and immunomodulation. Alternatively spliced transcript variants have been identified for this gene, including a CYP24A1 splice variant (CYP24sv) specifically-expressed in macrophage (Ren et al., 2005), which skips exons 1 and 2. The 372 amino acid variant protein (~43 kDa) lacks the

N-terminus, helices A’, A, B and B’ and portions of the β1 sheet, but likely retains the ability to bind heme and substrate in some capacity. Ren and co-authors concluded that CYP24sv represents a cytosolic variant with dominant negative function that quarantines 25-hydroxyvitamin

D3 and other hormonal forms of vitamin D, slowing the rate of their metabolism in the mitochondria or ER. Structural analysis of CYP24A1 (PDB: 3K9V) suggests that CYP24sv’s 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 via interactions with helices F-G and the β1 and β4 sheets systems. It is notable that exon 1 and 2 exclusion in CYP24A1 is exacerbated in human tumors of the breast and colon, where N-terminally-truncated splice

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 variants of approximately 40, 42, and 44 kDa have been identified (Fischer et al., 2009; Horváth et al., 2010; Scheible et al., 2014). An additional, prostate cancer-related, splice variant of

CYP24A1 that cleanly skips exon 10 has also been described (Horvath et al., 2010; Muindi et al.,

2007). Exon 10 encodes much of the proteins proximal surface, including the β1-3 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 redox partner binding, giving rise to an alternative dominant negative form that may retain membrane binding features; loss of exon Downloaded from 10 could also refine the alternative functions of cytosolic splice variants lacking exons 1 and 2.

Figure 4. Endo-xenobiotic Crosstalk among Cytochrome P450 and Nuclear Receptor dmd.aspetjournals.org

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 at ASPET Journals on September 30, 2021 genes participate in phase I detoxification of many of these compounds, including model substrates Benzo[a]pyrene (via CYP3A4) and Calcifediol (via CYP24A1). CYP genes are classically induced to silence endo-xenobiotic 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 CYPs in a given tissue, however, because each gene is sensitive to an infinite number of environmentally-sensitive, alternative splicing events, each individual may express a unique, tissue-specific “CYP gene cloud” comprised of both wild-type (WT) and splice-altered variant forms (e.g. SV1, SV2, etc.). CYP 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

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254 with alternative metabolic function or cellular role. When coupled with existing paradigms of alternate CYP trafficking and membrane-associated cooperativity, an integrated network of crosstalk among 57 CYP and 48 NR genes begins to emerge, as novel CYP metabolites may engage

NR signaling pathways in unique ways that re-program 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 adapting to cellular stress created by one’s unique personal history and disease-status. Downloaded from

dmd.aspetjournals.org

at ASPET Journals on September 30, 2021

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

TABLES:

TABLE 1. Pathologies Associated with CYP Splice Variants

CYP Related Variant Form Reference Isoform Disease Chinta et al. (2005) J Neurochem 93(3):724-736 CYP1A1 ∆Exon 2 and 6 Ovarian Cancer Bauer et al. (2007) Int J Oncol 31:211-218 Leung et al. (2005) Cancer Res 65:3726-3734 Stoilov et al. (1998) Am J Hum Genet 62:573- Partial ∆Exon CYP1B1 Glaucoma 584 Downloaded from 2 Li et al. (2014) J Proteome Res 13(5):2679-2687 CYP2A6 Deletions Lung Cancer Liu et al. (2013) Tumour Biol 34(5):2651-7. Liver/Colon Miles et al. (1989) Nucl Acids Res 17(20):8241- CYP2B6 ∆Exons 4-6 dmd.aspetjournals.org Cancer 8255 Nakayama et al. (2002) BBRC 297(5):1135-1139 ∆Exons 2 and Essential CYP8A1 Nakayama et al. (2002) Am Heart J 143(5):797- 9 Hypertension 801 at ASPET Journals on September 30, 2021 Congenital Trans-splicing Hampf et al. (2001) Clin Endocrinol Metab CYP11B2 Adrenal to CYP11B1 86(9):4445-4452 Hyperplasia 17-alpha Yamaguchi et al. (1998) Hum Genet 102(6):635- Hydroxylase 639 ∆Exons 2, 4, 6 Deficiency, CYP17A1 Costa-Santos et al. (2004) Clin Endocrinol Metab or 8 Congenital 89(1):43-48 Adrenal Hwang et al. (2011) PLoS One 6(9):e25492 Hyperplasia Aromatase CYP19A1 ∆Exon 5 Pepe et al. (2007) Clin Endocrinol 67(5):698-705 Deficiency Breast, Colon, Muindini et al (2007) Steroid Biochem Mol Biol CYP24A1 ∆Exons 9-11 and Prostate 103(3-5):334-337 Cancer Atherosclerosis Elmabsout et al. (2012) PLoS One 7(5):e36839 CYP26B1 ∆Exon 2 Oral Cancer Chen et al. (2014) Sci World J 2014:810561

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Garuti et al. (1996) J Lipid Res 37(7):1459-1467 Cererotendinous Verrips et al. (1997) Hu Genet 100(2):284-286 CYP27A1 ∆Exons 4,6, 7 Xanthomatosis Chen et al. (1998) Biochemistry 37(43):15050- (CTX) 15056 Maas et al. (2001) Clin Canc Res 7(4):868-875 Flanagan et al. (2003) Rec Res Canc Res Glioblastoma, 164:157-167 Leukemia, Fischer et al. (2007) Canc Genom Proteom CYP27B1 ∆Exons 4-7 Melanoma, 4(4):295 Downloaded from Cervical Cancer, Wu et al. (2007) Endocrinology 148(7):3410- Ovarian Cancer 3418 Fischer et al. (2009) Anticanc Res 29(9):3627-

3633 dmd.aspetjournals.org Alzheimer’s Li et al (2013) Neurosci Lett 549:18-23 CYP46A1 Intron 2 Disease Li et al (2013) J Neurol 260(7):1701-1708

at ASPET Journals on September 30, 2021

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Table 2. Summary of Human CYP Splice Variants

Ratio of Enzyme Transcript Mol. Total # of Total # Amino Disease Viable Family Size Exons Weight Variant of Acids Links Protein (Gene #) (nucleotides) (kDa) RNAs SNPs (Total)

CYP1 2683- 512- 58.2- 0.64‡ 3-7 49† 1052 203 (3) 5988 543 60.8 (31) Downloaded from

CYP2 1681- 491- 55.7- 0.50 5-9 281 11853 413 (16) 5024 596 67.6 (141)

CYP3 1604- 502- 57.1- 0.47 13-14 120 3029 93 dmd.aspetjournals.org (4) 2167 534 61.3 (56)

CYP4 1678- 505- 59.1- 0.53 5-16 170 9725 37 (12) 3395 638 71.3 (90)

CYP5 0.48 at ASPET Journals on September 30, 2021 2093 13 594 66.9 30 5274 6 (1) (14) CYP7 2765- 504- 57.7- 0.80 6 4 3688 35 (2) 2950 506 58.3 (3) CYP8 3974- 501- 58.1- 0.77 1-10 9 1752 16 (2) 5632 527 59.5 (7) CYP11 2311- 503- 57.6- 0.57 9 52 1807 74 (3) 3558 521 60.1 (30) CYP17 0.57 2001 8 508 57.4 13 179 31 (1) (7) CYP19 0.57 4479 10 503 57.9 47 2178 45 (1) (27) CYP20 0.54 1811 13 470 53.4 21 1308 1 (1) (11) CYP21 0.52 2153 10 495 56.0 30 91 35 (1) (16)

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

CYP24 0.46 3266 12 514 58.9 17 711 19 (1) (8) CYP26 1569- 497- 56.2- 0.68 6-7 23 858 22 (3) 4536 522 57.5 (16) CYP27 2579- 372- 42.6- 0.58 8-9 55 1582 58 (3) 5117 531 60.2 (32) CYP39 0.50 2623 12 469 54.1 15 2179 2 (1) (8) CYP46 0.57 2186 15 500 56.8 11 952 7 Downloaded from (1) (6) CYP51 0.65 2817 6 860 98.0 18 388 2 (1) (12)

dmd.aspetjournals.org Total 1569- 372- 42.6- 1-16 965 48,384 1097 0.534 57 5632 860 98.0 (515) † Total Number of Variant RNA Transcripts represents the total of all non-redundant transcripts identified for human CYP forms in the AceView, ENSEMBL and Pubmed database. Transcripts with the same predicted protein molecular weight (in kilodaltons (kDa)) with a less than 4 base pair difference in nucleotide transcript length were considered the at ASPET Journals on September 30, 2021 same transcript. Data for SNPs was obtained from GeneCards.org, and Disease Links were obtained from the MalaCards.org.

‡ Ratio of Viable Protein represents the fraction of human CYP 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.

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Downloaded from dmd.aspetjournals.org at ASPET Journals on September 30, 2021

DMD Fast Forward. Published on February 10, 2017 as DOI: 10.1124/dmd.116.073254 This article has not been copyedited and formatted. The final version may differ from this version.

DMD # 73254

Downloaded from dmd.aspetjournals.org

at ASPET Journals on September 30, 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 cofactor 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 base pair 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.