Cigarette Smoking-Associated Isoform Switching and 3’ UTR Lengthening Via Alternative Polyadenylation

Background Cigarette smoking accounts for approximately one in five deaths in the United States. Previous genomic studies have primarily focused on gene level differential expression to identify related molecular signatures and pathways, but the genome-wide effects of smoking on alternative isoform regulation and posttranscriptional modulation have not yet been described. Results We conducted RNA sequencing (RNA-seq) in whole-blood samples of 454 current and 767 former smokers in COPDGene Study. We assessed the association of current smoking with differential expression of genes and isoforms and differential usage of isoforms and exons. At 10% FDR, we detected 3,167 differentially expressed genes, 2,014 differentially expressed isoforms, 945 differentially used isoforms and 160 differentially used exons. Genes containing differentially used isoforms were enriched in biological pathways involving GTPase activity and innate immunity. The majority of these genes were not differentially expressed, thus not identifiable from conventional differential gene expression analysis. Isoform switch analysis revealed for the first time widespread 3′ UTR lengthening associated with cigarette smoking, where current smokers were found to have higher expression and usage of isoforms with markedly longer 3′ UTRs. The lengthening of 3′ UTRs appears to be mediated through alternative usage of distal polyadenylation sites, and these extended 3′ UTR regions are significantly enriched with functional sequence elements including adenylate-uridylate (AU)-rich elements, microRNA and RNA-protein binding sites. Expression quantitative trait locus analyses on differentially used 3′ UTRs identified 79 known GWAS variants associated with multiple smoking-related human diseases and traits. Conclusions Smoking elicits widespread transcriptional and posttranscriptional alterations with disease implications. It induces alternative polyadenylation (APA) events resulting in a switch towards the usage of isoforms with strikingly longer 3′ UTRs in genes related to multiple biological pathways including GTPase activity and innate immunity. The extended 3′ UTR regions are enriched with functional sequence elements facilitating post-transcriptional regulation of protein expression and mRNA stability. These findings warrant further studies on APA events as potential biomarkers and novel therapeutic targets for smoking-related diseases. ### Competing Interest Statement P. Castaldi has received personal fees and grant support from GlaxoSmithKline, Bayer, and Novartis. C. Hersh has received grants from NHLBI, Bayer, Boehringer-Ingelheim, Novartis and Vertex. A. Laederach has received consultant fees from Ribometrix. ### Clinical Trial NCT00608764 ### Funding Statement This work was funded by R01 HL124233, R01 HL147326, R01 HL111527, U01 HL089897, U01 HL089856, R01HL125583, R01HL130512, R01 GM101237, R01 HL11152, K25HL140186 and K08HL141601. Research reported in this publication was supported by the NHLBI, NIGMS and FDA Center for Tobacco Products (CTP). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the Food and Drug Administration. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Brigham and Womens HospitalPartners Human Research Committee2007P000554 All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Data have been deposited in GEO.