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Alternative Splicing in the Cytochrome P450 Superfamily Expands Protein Diversity To 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 1 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 2 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 3 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. 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 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 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 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
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