Post transcriptional modifications of RNA are powerful mechanisms by which eukaryotes expand their genetic diversity. For instance, researchers estimate that most transcripts in humans undergo alternative splicing and alternative polyadenylation. These splicing events produce distinct RNA molecules, which in turn yield distinct protein isoforms and/or influence RNA stability, translation, nuclear export, and RNA/protein cellular localization. Due to their pervasiveness and impact, we hypothesized that alternative splicing and alternative polyadenylation in brain can contribute to a predisposition for voluntary alcohol consumption. Using the HXB/BXH recombinant inbred rat panel (a subset of the Hybrid Rat Diversity Panel), we generated over one terabyte of brain RNA sequencing data (total RNA) and identified novel splice variants (via StringTie) and alternative polyadenylation sites (via aptardi) to determine the transcriptional landscape in the brains of these animals. After establishing an analysis pipeline to ascertain high quality transcripts, we quantitated transcripts and integrated genotype data to identify candidate transcript coexpression networks and individual candidate transcripts associated with predisposition to voluntary alcohol consumption in the two-bottle choice paradigm. For genes that were previously associated with this trait (e.g., Lrap, Ift81, and P2rx4) (Saba et al., Febs. J., 282, 3556-3578, Saba et al., Genes. Brain. Behav., 20, e12698), we were able to distinguish between transcript variants to provide further information about the specific isoforms related to the trait. We also identified additional candidate transcripts associated with the trait of voluntary alcohol consumption (i.e., isoforms of Mapkapk5, Aldh1a7, and Map3k7). Consistent with our previous work, our results indicate that transcripts and networks related to inflammation and the immune system in brain can be linked to voluntary alcohol consumption. Overall, we have established a pipeline for including the quantitation of alternative splicing and alternative polyadenylation variants in the transcriptome in the analysis of the relationship between the transcriptome and complex traits.

Department of Pharmaceutical Sciences University of Colorado Anschutz Medical Campus Aurora CO United States

Department of Pharmacology University of Colorado Anschutz Medical Campus Aurora CO United States

Institute of Physiology of the Czech Academy of Sciences Prague Czechia

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Abiola O., Angel J. M., Avner P., Bachmanov A. A., Belknap J. K., Bennett B., et al. (2003). The Nature and Identification of Quantitative Trait Loci: a Community's View. Nat. Rev. Genet. 4, 911–916. 10.1038/nrg1206 PubMed DOI PMC

Arlinde C., Sommer W., Björk K., Reimers M., Hyytiä P., Kiianmaa K., et al. (2004). A Cluster of Differentially Expressed Signal Transduction Genes Identified by Microarray Analysis in a Rat Genetic Model of Alcoholism. Pharmacogenomics J. 4, 208–218. 10.1038/sj.tpj.6500243 PubMed DOI

Asatryan L., Popova M., Perkins D., Trudell J. R., Alkana R. L., Davies D. L. (2010). Ivermectin Antagonizes Ethanol Inhibition in Purinergic P2X4 Receptors. J. Pharmacol. Exp. Ther. 334, 720–728. 10.1124/jpet.110.167908 PubMed DOI PMC

Baik I., Cho N. H., Kim S. H., Han B.-G., Shin C. (2011). Genome-wide Association Studies Identify Genetic Loci Related to Alcohol Consumption in Korean Men. Am. J. Clin. Nutr. 93, 809–816. 10.3945/ajcn.110.001776 PubMed DOI

Beaudoing E., Gautheret D. (2001). Identification of Alternate Polyadenylation Sites and Analysis of Their Tissue Distribution Using EST Data. Genome Res. 11, 1520–1526. 10.1101/gr.190501 PubMed DOI PMC

Bhogaraju S., Cajanek L., Fort C., Blisnick T., Weber K., Taschner M., et al. (2013). Molecular Basis of Tubulin Transport within the Cilium by IFT74 and IFT81. Science 341, 1009–1012. 10.1126/science.1240985 PubMed DOI PMC

Black W. J., Stagos D., Marchitti S. A., Nebert D. W., Tipton K. F., Bairoch A., et al. (2009). Human Aldehyde Dehydrogenase Genes: Alternatively Spliced Transcriptional Variants and Their Suggested Nomenclature. Pharmacogenet Genomics 19, 893–902. 10.1097/FPC.0b013e3283329023 PubMed DOI PMC

Broman K. W., Sen S. (2009). A Guide to QTL Mapping with R/qtl. New York: Springer-Verlag. 10.1007/978-0-387-92125-9 DOI

Bullard J. H., Purdom E., Hansen K. D., Dudoit S. (2010). Evaluation of Statistical Methods for Normalization and Differential Expression in mRNA-Seq Experiments. BMC Bioinformatics 11, 94. 10.1186/1471-2105-11-94 PubMed DOI PMC

Cargnello M., Roux P. P. (2011). Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases. Microbiol. Mol. Biol. Rev. 75, 50–83. 10.1128/MMBR.00031-10 PubMed DOI PMC

Churchill G. A., Doerge R. W. (1994). Empirical Threshold Values for Quantitative Trait Mapping. Genetics 138, 963–971. 10.1093/genetics/138.3.963 PubMed DOI PMC

Dafne Giammartino D. G., Nishida K., Manley J. L. (2011). Mechanisms and Consequences of Alternative Polyadenylation. Mol. Cel 43, 853–866. 10.1016/j.molcel.2011.08.017 PubMed DOI PMC

Davies D. L., Machu T. K., Guo Y., Alkana R. L. (2002). Ethanol Sensitivity in ATP-Gated P2X Receptors Is Subunit Dependent. Alcohol. Clin. Exp. Res. 26, 773–778. 10.1111/j.1530-0277.2002.tb02604.x PubMed DOI

Dawson D. A., Grant B. F. (2011). The "Gray Area" of Consumption between Moderate and Risk Drinking*. J. Stud. Alcohol. Drugs 72, 453–458. 10.15288/jsad.2011.72.453 PubMed DOI PMC

DePaula-Silva A. B., Gorbea C., Doty D. J., Libbey J. E., Sanchez J. M. S., Hanak T. J., et al. (2019). Differential Transcriptional Profiles Identify Microglial- and Macrophage-specific Gene Markers Expressed during Virus-Induced Neuroinflammation. J. Neuroinflammation 16, 152. 10.1186/s12974-019-1545-x PubMed DOI PMC

Derti A., Garrett-Engele P., Macisaac K. D., Stevens R. C., Sriram S., Chen R., et al. (2012). A Quantitative Atlas of Polyadenylation in Five Mammals. Genome Res. 22, 1173–1183. 10.1101/gr.132563.111 PubMed DOI PMC

Dredge B. K., Polydorides A. D., Darnell R. B. (2001). The Splice of Life: Alternative Splicing and Neurological Disease. Nat. Rev. Neurosci. 2, 43–50. 10.1038/35049061 PubMed DOI

Faustino N. A., Cooper T. A. (2003). Pre-mRNA Splicing and Human Disease. Genes Dev. 17, 419–437. 10.1101/gad.1048803 PubMed DOI

Garcia-Blanco M. A., Baraniak A. P., Lasda E. L. (2004). Alternative Splicing in Disease and Therapy. Nat. Biotechnol. 22, 535–546. 10.1038/nbt964 PubMed DOI

Gibbs R. A., Weinstock G. M., Metzker M. L., Muzny D. M., Sodergren E. J., Scherer S., et al. (2004). Genome Sequence of the Brown Norway Rat Yields Insights into Mammalian Evolution. Nature 428, 493–521. 10.1038/nature02426 PubMed DOI

Gofman L., Cenna J. M., Potula R. (2014). P2X4 Receptor Regulates Alcohol-Induced Responses in Microglia. J. Neuroimmune Pharmacol. 9, 668–678. 10.1007/s11481-014-9559-8 PubMed DOI PMC

Grant J. D., Agrawal A., Bucholz K. K., Madden P. A. F., Pergadia M. L., Nelson E. C., et al. (2009). Alcohol Consumption Indices of Genetic Risk for Alcohol Dependence. Biol. Psychiatry 66, 795–800. 10.1016/j.biopsych.2009.05.018 PubMed DOI PMC

Harrall K. K., Kechris K. J., Tabakoff B., Hoffman P. L., Hines L. M., Tsukamoto H., et al. (2016). Uncovering the Liver's Role in Immunity through RNA Co-expression Networks. Mamm. Genome 27, 469–484. 10.1007/s00335-016-9656-5 PubMed DOI PMC

Hartley C. A., McKenna M. C., Salman R., Holmes A., Casey B. J., Phelps E. A., et al. (2012). Serotonin Transporter Polyadenylation Polymorphism Modulates the Retention of Fear Extinction Memory. Proc. Natl. Acad. Sci. 109, 5493–5498. 10.1073/pnas.1202044109 PubMed DOI PMC

Havlak P., Chen R., Durbin K. J., Egan A., Ren Y., Song X.-Z., et al. (2004). The Atlas Genome Assembly System. Genome Res. 14, 721–732. 10.1101/gr.2264004 PubMed DOI PMC

Hermsen R., de Ligt J., Spee W., Blokzijl F., Schäfer S., Adami E., et al. (2015). Genomic Landscape of Rat Strain and Substrain Variation. BMC Genomics 16, 357. 10.1186/s12864-015-1594-1 PubMed DOI PMC

Hoffman P. L., Saba L. M., Flink S., Grahame N. J., Kechris K., Tabakoff B. (2014). Genetics of Gene Expression Characterizes Response to Selective Breeding for Alcohol Preference. Genes, Brain Behav. 13, 743–757. 10.1111/gbb.12175 PubMed DOI PMC

Hong M., Zhukareva V., Vogelsberg-Ragaglia V., Wszolek Z., Reed L., Miller B. I., et al. (1998). Mutation-specific Functional Impairments in Distinct Tau Isoforms of Hereditary FTDP-17. Science 282, 1914–1917. 10.1126/science.282.5395.1914 PubMed DOI

Hoque M., Ji Z., Zheng D., Luo W., Li W., You B., et al. (2013). Analysis of Alternative Cleavage and Polyadenylation by 3′ Region Extraction and Deep Sequencing. Nat. Methods 10, 133–139. 10.1038/nmeth.2288 PubMed DOI PMC

Hutton M., Lendon C. L., Rizzu P., Baker M., Froelich S., Houlden H., et al. (1998). Association of Missense and 5′-Splice-Site Mutations in Tau with the Inherited Dementia FTDP-17. Nature 393, 702–705. 10.1038/31508 PubMed DOI

Jackson B., Brocker C., Thompson D. C., Black W., Vasiliou K., Nebert D. W., et al. (2011). Update on the Aldehyde Dehydrogenase Gene (ALDH) Superfamily. Hum. Genomics 5, 283–303. 10.1186/1479-7364-5-4-283 PubMed DOI PMC

Ji X., Li P., Fuscoe J. C., Chen G., Xiao W., Shi L., et al. (2020). A Comprehensive Rat Transcriptome Built from Large Scale RNA-Seq-Based Annotation. Nucleic Acids Res. 48, 8320–8331. 10.1093/nar/gkaa638 PubMed DOI PMC

Johnson W. E., Li C., Rabinovic A. (2007). Adjusting Batch Effects in Microarray Expression Data Using Empirical Bayes Methods. Biostatistics 8, 118–127. 10.1093/biostatistics/kxj037 PubMed DOI

Kalyana-Sundaram S., Kumar-Sinha C., Shankar S., Robinson D. R., Wu Y.-M., Cao X., et al. (2012). Expressed Pseudogenes in the Transcriptional Landscape of Human Cancers. Cell 149, 1622–1634. 10.1016/j.cell.2012.04.041 PubMed DOI PMC

Karunakaran K. B., Chaparala S., Lo C. W., Ganapathiraju M. K. (2020). Cilia Interactome with Predicted Protein-Protein Interactions Reveals Connections to Alzheimer's Disease, Aging and Other Neuropsychiatric Processes. Sci. Rep. 10, 15629. 10.1038/s41598-020-72024-4 PubMed DOI PMC

Kawasawa Y. I., Mohammad S., Son A. I., Morizono H., Basha A., Salzberg A. C., et al. (2017). Genome-wide Profiling of Differentially Spliced mRNAs in Human Fetal Cortical Tissue Exposed to Alcohol. Alcohol 62, 1–9. 10.1016/j.alcohol.2017.05.001 PubMed DOI PMC

Kelemen O., Convertini P., Zhang Z., Wen Y., Shen M., Falaleeva M., et al. (2013). Function of Alternative Splicing. Gene 514, 1–30. 10.1016/j.gene.2012.07.083 PubMed DOI PMC

Kent W. J. (2002). BLAT-the BLAST-like Alignment Tool. Genome Res. 12, 656–664. 10.1101/gr.229202 PubMed DOI PMC

Kent W. J., Sugnet C. W., Furey T. S., Roskin K. M., Pringle T. H., Zahler A. M., et al. (2002). The Human Genome Browser at UCSC. Genome Res. 12, 996–1006. 10.1101/gr.229102 PubMed DOI PMC

Kim D., Langmead B., Salzberg S. L. (2015). HISAT: a Fast Spliced Aligner with Low Memory Requirements. Nat. Methods 12, 357–360. 10.1038/nmeth.3317 PubMed DOI PMC

Kimpel M. W., Strother W. N., McClintick J. N., Carr L. G., Liang T., Edenberg H. J., et al. (2007). Functional Gene Expression Differences between Inbred Alcohol-Preferring and -Non-Preferring Rats in Five Brain Regions. Alcohol 41, 95–132. 10.1016/j.alcohol.2007.03.003 PubMed DOI PMC

Kosten T. A. (2011). Pharmacologically Targeting the P2rx4 Gene on Maintenance and Reinstatement of Alcohol Self-Administration in Rats. Pharmacol. Biochem. Behav. 98, 533–538. 10.1016/j.pbb.2011.02.026 PubMed DOI PMC

Lalo U., Verkhratsky A., Pankratov Y. (2007). Ivermectin Potentiates ATP-Induced Ion Currents in Cortical Neurones: Evidence for Functional Expression of P2X4 Receptors? Neurosci. Lett. 421, 158–162. 10.1016/j.neulet.2007.03.078 PubMed DOI

Lander E., Kruglyak L. (1995). Genetic Dissection of Complex Traits: Guidelines for Interpreting and Reporting Linkage Results. Nat. Genet. 11, 241–247. 10.1038/ng1195-241 PubMed DOI

Langfelder P., Horvath S. (2008). WGCNA: an R Package for Weighted Correlation Network Analysis. BMC Bioinformatics 9, 559. 10.1186/1471-2105-9-559 PubMed DOI PMC

Langmead B., Salzberg S. L. (2012). Fast Gapped-Read Alignment with Bowtie 2. Nat. Methods 9, 357–359. 10.1038/nmeth.1923 PubMed DOI PMC

Lau A. G., Irier H. A., Gu J., Tian D., Ku L., Liu G., et al. (2010). Distinct 3'UTRs Differentially Regulate Activity-dependent Translation of Brain-Derived Neurotrophic Factor (BDNF). Proc. Natl. Acad. Sci. 107, 15945–15950. 10.1073/pnas.1002929107 PubMed DOI PMC

Lee W.-B., Choi W. Y., Lee D.-H., Shim H., Kim-Ha J., Kim Y.-J. (2019). OAS1 and OAS3 Negatively Regulate the Expression of Chemokines and Interferon-Responsive Genes in Human Macrophages. BMB Rep. 52, 133–138. 10.5483/bmbrep.2019.52.2.129 PubMed DOI PMC

Leek J. T., Johnson W. E., Parker H. S., Jaffe A. E., Storey J. D. (2012). The Sva Package for Removing Batch Effects and Other Unwanted Variation in High-Throughput Experiments. Bioinformatics 28, 882–883. 10.1093/bioinformatics/bts034 PubMed DOI PMC

Li B., Dewey C. N. (2011). RSEM: Accurate Transcript Quantification from RNA-Seq Data with or without a Reference Genome. BMC Bioinformatics 12, 323. 10.1186/1471-2105-12-323 PubMed DOI PMC

Li C., Xiong K., Weight F. F. (2000). Ethanol Inhibition of Adenosine 5′-Triphosphate-Activated Current in Freshly Isolated Adult Rat Hippocampal CA1 Neurons. Neurosci. Lett. 295, 77–80. 10.1016/s0304-3940(00)01586-x PubMed DOI

Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., et al. (2009). The Sequence Alignment/Map Format and SAMtools. Bioinformatics 25, 2078–2079. 10.1093/bioinformatics/btp352 PubMed DOI PMC

Li H. (2018). Minimap2: Pairwise Alignment for Nucleotide Sequences. Bioinformatics 34, 3094–3100. 10.1093/bioinformatics/bty191 PubMed DOI PMC

L. Koles L., S. Furst S., P. Illes P. (2007). Purine Ionotropic (P2X) Receptors. Cpd 13, 2368–2384. 10.2174/138161207781368747 PubMed DOI

Lo U., Selvaraj V., Plane J. M., Chechneva O. V., Otsu K., Deng W. (2014). p38α (MAPK14) Critically Regulates the Immunological Response and the Production of Specific Cytokines and Chemokines in Astrocytes. Sci. Rep. 4, 7405. 10.1038/srep07405 PubMed DOI PMC

Love M. I., Huber W., Anders S. (2014). Moderated Estimation of Fold Change and Dispersion for RNA-Seq Data with DESeq2. Genome Biol. 15, 550. 10.1186/s13059-014-0550-8 PubMed DOI PMC

Lusk R., Saba L. M., Vanderlinden L. A., Zidek V., Silhavy J., Pravenec M., et al. (2018). Unsupervised, Statistically Based Systems Biology Approach for Unraveling the Genetics of Complex Traits: A Demonstration with Ethanol Metabolism. Alcohol. Clin. Exp. Res. 42, 1177–1191. 10.1111/acer.13763 PubMed DOI PMC

Lusk R., Stene E., Banaei-Kashani F., Tabakoff B., Kechris K., Saba L. M. (2021). Aptardi Predicts Polyadenylation Sites in Sample-specific Transcriptomes Using High-Throughput RNA Sequencing and DNA Sequence. Nat. Commun. 12, 1652. 10.1038/s41467-021-21894-x PubMed DOI PMC

MacDonald C. C. (2019). Tissue‐specific Mechanisms of Alternative Polyadenylation: Testis, Brain, and beyond (2018 Update). WIREs RNA 10, e1526. 10.1002/wrna.1526 PubMed DOI PMC

Manning K. S., Cooper T. A. (2017). The Roles of RNA Processing in Translating Genotype to Phenotype. Nat. Rev. Mol. Cel Biol 18, 102–114. 10.1038/nrm.2016.139 PubMed DOI PMC

Marioni J. C., Mason C. E., Mane S. M., Stephens M., Gilad Y. (2008). RNA-seq: An Assessment of Technical Reproducibility and Comparison with Gene Expression Arrays. Genome Res. 18, 1509–1517. 10.1101/gr.079558.108 PubMed DOI PMC

Martin M. (2011). Cutadapt Removes Adapter Sequences from High-Throughput Sequencing Reads. EMBnet j. 17, 10–12. 10.14806/ej.17.1.200 DOI

Martín-Blanco E. (2000). p38 MAPK Signalling Cascades: Ancient Roles and New Functions. Bioessays 22, 637–645. 10.1002/1521-1878(200007)22:7<637::AID-BIES6>3.0.CO;2-E PubMed DOI

Martinez N. M., Lynch K. W. (2013). Control of Alternative Splicing in Immune Responses: many Regulators, many Predictions, Much Still to Learn. Immunol. Rev. 253, 216–236. 10.1111/imr.12047 PubMed DOI PMC

Martinez N. M., Pan Q., Cole B. S., Yarosh C. A., Babcock G. A., Heyd F., et al. (2012). Alternative Splicing Networks Regulated by Signaling in Human T Cells. RNA 18, 1029–1040. 10.1261/rna.032243.112 PubMed DOI PMC

Miura P., Sanfilippo P., Shenker S., Lai E. C. (2014). Alternative Polyadenylation in the Nervous System: To what Lengths Will 3′ UTR Extensions Take Us? Bioessays 36, 766–777. 10.1002/bies.201300174 PubMed DOI PMC

New L., Jiang Y., Zhao M., Liu K., Zhu W., Flood L. J., et al. (1998). PRAK, a Novel Protein Kinase Regulated by the P38 MAP Kinase. EMBO J. 17, 3372–3384. 10.1093/emboj/17.12.3372 PubMed DOI PMC

Nilsen T. W., Graveley B. R. (2010). Expansion of the Eukaryotic Proteome by Alternative Splicing. Nature 463, 457–463. 10.1038/nature08909 PubMed DOI PMC

Nissim-Rafinia M., Kerem B. (2002). Splicing Regulation as a Potential Genetic Modifier. Trends Genet. 18, 123–127. 10.1016/s0168-9525(01)02619-1 PubMed DOI

Oliveira L. d. M., Teixeira F. M. E., Sato M. N. (2018). Impact of Retinoic Acid on Immune Cells and Inflammatory Diseases. Mediators Inflamm. 2018, 1–17. 10.1155/2018/3067126 PubMed DOI PMC

Pan Q., Shai O., Lee L. J., Frey B. J., Blencowe B. J. (2008). Deep Surveying of Alternative Splicing Complexity in the Human Transcriptome by High-Throughput Sequencing. Nat. Genet. 40, 1413–1415. 10.1038/ng.259 PubMed DOI

Pertea M., Pertea G. M., Antonescu C. M., Chang T.-C., Mendell J. T., Salzberg S. L. (2015). StringTie Enables Improved Reconstruction of a Transcriptome from RNA-Seq Reads. Nat. Biotechnol. 33, 290–295. 10.1038/nbt.3122 PubMed DOI PMC

Petruccelli E., Brown T., Waterman A., Ledru N., Kaun K. R. (2020). Alcohol Causes Lasting Differential Transcription in Drosophila Mushroom Body Neurons. Genetics 215, 103–116. 10.1534/genetics.120.303101 PubMed DOI PMC

Pravenec M., Klír P., Kren V., Zicha J., Kunes J. (1989). An Analysis of Spontaneous Hypertension in Spontaneously Hypertensive Rats by Means of New Recombinant Inbred Strains. J. Hypertens. 7, 217–221. 10.1097/00004872-198903000-00008 PubMed DOI

Pravenec M., Saba L. M., Zídek V., Landa V., Mlejnek P., Šilhavý J., et al. (2018). Systems Genetic Analysis of Brown Adipose Tissue Function. Physiol. Genomics 50, 52–66. 10.1152/physiolgenomics.00091.2017 PubMed DOI PMC

Ren F., Zhang N., Zhang L., Miller E., Pu J. J. (2020). Alternative Polyadenylation: a New Frontier in post Transcriptional Regulation. Biomark Res. 8, 67. 10.1186/s40364-020-00249-6 PubMed DOI PMC

Rhinn H., Qiang L., Yamashita T., Rhee D., Zolin A., Vanti W., et al. (2012). Alternative α-synuclein Transcript Usage as a Convergent Mechanism in Parkinson's Disease Pathology. Nat. Commun. 3, 1084. 10.1038/ncomms2032 PubMed DOI PMC

Risso D., Schwartz K., Sherlock G., Dudoit S. (2011). GC-content Normalization for RNA-Seq Data. BMC Bioinformatics 12, 480. 10.1186/1471-2105-12-480 PubMed DOI PMC

Rodd Z. A., Bertsch B. A., Strother W. N., Le-Niculescu H., Balaraman Y., Hayden E., et al. (2007). Candidate Genes, Pathways and Mechanisms for Alcoholism: an Expanded Convergent Functional Genomics Approach. Pharmacogenomics J. 7, 222–256. 10.1038/sj.tpj.6500420 PubMed DOI

Saba L. M., Flink S. C., Vanderlinden L. A., Israel Y., Tampier L., Colombo G., et al. (2015). The Sequenced Rat Brain Transcriptome - its Use in Identifying Networks Predisposing Alcohol Consumption. Febs J. 282, 3556–3578. 10.1111/febs.13358 PubMed DOI PMC

Saba L. M., Hoffman P. L., Homanics G. E., Mahaffey S., Daulatabad S. V., Janga S. C., et al. (2020). A Long Non‐coding RNA ( Lrap ) Modulates Brain Gene Expression and Levels of Alcohol Consumption in Rats. Genes. Brain Behav. 20, e12698. 10.1111/gbb.12698 PubMed DOI PMC

Sanfilippo P., Wen J., Lai E. C. (2017). Landscape and Evolution of Tissue-specific Alternative Polyadenylation across Drosophila Species. Genome Biol. 18, 229. 10.1186/s13059-017-1358-0 PubMed DOI PMC

Sasabe T., Ishiura S. (2010). Alcoholism and Alternative Splicing of Candidate Genes. Ijerph 7, 1448–1466. 10.3390/ijerph7041448 PubMed DOI PMC

Sen Ś., Churchill G. A. (2001). A Statistical Framework for Quantitative Trait Mapping. Genetics 159, 371–387. 10.1093/genetics/159.1.371 PubMed DOI PMC

Shannon P., Markiel A., Ozier O., Baliga N. S., Wang J. T., Ramage D., et al. (2003). Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks. Genome Res. 13, 2498–2504. 10.1101/gr.1239303 PubMed DOI PMC

Singh S., Brocker C., Koppaka V., Chen Y., Jackson B. C., Matsumoto A., et al. (2013). Aldehyde Dehydrogenases in Cellular Responses to Oxidative/electrophilicstress. Free Radic. Biol. Med. 56, 89–101. 10.1016/j.freeradbiomed.2012.11.010 PubMed DOI PMC

Smit A., Hubley R., Green P. (1996). RepeatMasker‬ Open-3.01996. Available at: http://www.repeatmasker.org (Accessed November‬‬‬‬‬ 22, 2018).

Sommer W., Arlinde C., Heilig M. (2005). The Search for Candidate Genes of Alcoholism: Evidence from Expression Profiling Studies. Addict. Biol. 10, 71–79. 10.1080/13556210412331327821 PubMed DOI

Spillantini M. G., Murrell J. R., Goedert M., Farlow M. R., Klug A., Ghetti B. (1998). Mutation in the Tau Gene in Familial Multiple System Tauopathy with Presenile Dementia. Proc. Natl. Acad. Sci. 95, 7737–7741. 10.1073/pnas.95.13.7737 PubMed DOI PMC

STAR Consortium (2008). SNP and Haplotype Mapping for Genetic Analysis in the Rat. Nat. Genet. 40, 560–566. 10.1038/ng.124 PubMed DOI PMC

Tabakoff B., Saba L., Saba L., Printz M., Flodman P., Hodgkinson C., et al. (2009). Genetical Genomic Determinants of Alcohol Consumption in Rats and Humans. BMC Biol. 7, 70. 10.1186/1741-7007-7-70 PubMed DOI PMC

Tabakoff B., Smith H., Vanderlinden L. A., Hoffman P. L., Saba L. M. (2019). Networking in Biology: The Hybrid Rat Diversity Panel. Methods Mol. Biol. 2018, 213–231. 10.1007/978-1-4939-9581-3_10 PubMed DOI

Tian B., Manley J. L. (2017). Alternative Polyadenylation of mRNA Precursors. Nat. Rev. Mol. Cel Biol 18, 18–30. 10.1038/nrm.2016.116 PubMed DOI PMC

Touloupi K., Küblbeck J., Magklara A., Molnár F., Reinisalo M., Konstandi M., et al. (2019). The Basis for Strain-dependent Rat Aldehyde Dehydrogenase 1A7 (ALDH1A7) Gene Expression. Mol. Pharmacol. 96, 655–663. 10.1124/mol.119.117424 PubMed DOI

Van Booven D., Mengying Linull, Sunil Rao J., Sunil Rao J., Blokhin I. O., Dayne Mayfield R., Barbier E., et al. (2021). Alcohol Use Disorder Causes Global Changes in Splicing in the Human Brain. Transl Psychiatry 11, 2. 10.1038/s41398-020-01163-z PubMed DOI PMC

Vanderlinden L. A., Saba L. M., Printz M. P., Flodman P., Koob G., Richardson H. N., et al. (2014). Is the Alcohol Deprivation Effect Genetically Mediated? Studies with HXB/BXH Recombinant Inbred Rat Strains. Alcohol. Clin. Exp. Res. 38, 2148–2157. 10.1111/acer.12471 PubMed DOI PMC

Vanin E. F. (1985). Processed Pseudogenes: Characteristics and Evolution. Annu. Rev. Genet. 19, 253–272. 10.1146/annurev.ge.19.120185.001345 PubMed DOI

Verhulst B., Neale M. C., Kendler K. S. (2015). The Heritability of Alcohol Use Disorders: a Meta-Analysis of Twin and Adoption Studies. Psychol. Med. 45, 1061–1072. 10.1017/S0033291714002165 PubMed DOI PMC

Wang E. T., Sandberg R., Luo S., Khrebtukova I., Zhang L., Mayr C., et al. (2008). Alternative Isoform Regulation in Human Tissue Transcriptomes. Nature 456, 470–476. 10.1038/nature07509 PubMed DOI PMC

Wang G.-S., Cooper T. A. (2007). Splicing in Disease: Disruption of the Splicing Code and the Decoding Machinery. Nat. Rev. Genet. 8, 749–761. 10.1038/nrg2164 PubMed DOI

Winkler A., Mahal B., Kiianmaa K., Zieglgänsberger W., Spanagel R. (1999). Effects of Chronic Alcohol Consumption on the Expression of Different NR1 Splice Variants in the Brain of AA and ANA Lines of Rats. Mol. Brain Res. 72, 166–175. 10.1016/s0169-328x(99)00218-1 PubMed DOI

Xiao C., Zhou C., Li K., Davies D. L., Ye J. H. (2008). Purinergic Type 2 Receptors at GABAergic Synapses on Ventral Tegmental Area Dopamine Neurons Are Targets for Ethanol Action. J. Pharmacol. Exp. Ther. 327, 196–205. 10.1124/jpet.108.139766 PubMed DOI PMC

Yates A. D., Achuthan P., Akanni W., Allen J., Allen J., Alvarez-Jarreta J., et al. (2020). Ensembl 2020. Nucleic Acids Res. 48, D682–D688. 10.1093/nar/gkz966 PubMed DOI PMC

Yeh H.-S., Yong J. (2016). Alternative Polyadenylation of mRNAs: 3′-Untranslated Region Matters in Gene Expression. Mol. Cell 39, 281–285. 10.14348/molcells.2016.0035 PubMed DOI PMC

Yoon O. K., Hsu T. Y., Im J. H., Brem R. B. (2012). Genetics and Regulatory Impact of Alternative Polyadenylation in Human B-Lymphoblastoid Cells. Plos Genet. 8, e1002882. 10.1371/journal.pgen.1002882 PubMed DOI PMC

Yoon Y., McKenna M. C., Rollins D. A., Song M., Nuriel T., Gross S. S., et al. (2013). Anxiety-associated Alternative Polyadenylation of the Serotonin Transporter mRNA Confers Translational Regulation by hnRNPK. Proc. Natl. Acad. Sci. 110, 11624–11629. 10.1073/pnas.1301485110 PubMed DOI PMC

Zhang B., Horvath S. (2005). A General Framework for Weighted Gene Co-expression Network Analysis. Stat. Appl. Genet. Mol. Biol. 4, Article17. 10.2202/1544-6115.1128 PubMed DOI

Zhang H., Lee J., Tian B. (2005). Biased Alternative Polyadenylation in Human Tissues. Genome Biol. 6, R100. 10.1186/gb-2005-6-12-r100 PubMed DOI PMC