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

Abstract

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. 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-prime UTR lengthening associated with cigarette smoking, where current smokers were found to have higher expression and usage of isoforms with markedly longer 3-prime UTRs. The lengthening of 3-prime UTRs appears to be mediated through alternative usage of distal polyadenylation sites, and these extended 3-prime 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-prime UTRs identified 79 known GWAS variants associated with multiple smoking-related human diseases and traits. 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-prime UTRs in genes related to multiple biological pathways including GTPase activity and innate immunity. The extended 3-prime 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.

Identification of Smoking‐Associated Transcriptome Aberration in Blood with Machine Learning Methods

“…Data. The RNA-seq data in whole-blood samples on 454 current and 767 former smokers were accessed from the Gene Expression Omnibus (GEO) database under the accession number GSE171730 [8]. We separated the samples into two classes based on their smoking history: current smokers and former smokers.…”

“…Therefore, gene expression alterations are important for smoking response. Considering that alternative splicing is applied by up to 95% of human genes for producing proteins with different functions, a recent study has focused on identifying smoking-associated isoforms and found that 3 ′ UTR lengthening was widely associated with cigarette smoking [7,8]. A total of 945 differentially expressed isoforms were identified in this study by using the classic statistic method.…”

“…In the present study, we applied the Boruta [10] and four feature ranking algorithms to identify the isoforms with important roles in distinguishing smokers from former smokers based on the expression data of 85,437 isoforms on current and former smokers collected in the previous study [8]. The incremental feature selection (IFS) method [11] was employed to further analyze the results yielded by above methods for extracting optimal features, building efficient classification models, and interesting classification rules.…”