Identification of Splicing Quantitative Trait Loci (sQTL) in Drosophila melanogaster with Developmental Lead (Pb2+) Exposure

Abstract

Lead (Pb) poisoning has been a major public health issue globally and the recent Flint water crisis has drawn nation-wide attention to its effects. To better understand how lead plays a role as a neurotoxin, we utilized the Drosophila melanogaster model to study the genetic effects of lead exposure during development and identified lead-responsive genes. In our previous studies, we have successfully identified hundreds of lead-responsive expression QTLs (eQTLs) by using RNA-seq analysis on heads collected from the Drosophila Synthetic Population Resource. Cis-eQTLs, also known as allele-specific expression (ASE) polymorphisms, are generally single-nucleotide polymorphisms in the promoter regions of genes that affect expression of the gene, such as by inhibiting the binding of transcription factors. Trans-eQTLs are genes that regulate mRNA levels for many genes, and are generally thought to be SNPs in trans-acting transcription or translation factors. In this study, we focused our attention on alternative splicing events that are affected by lead exposure. Splicing QTLs (sQTLs), which can be caused by SNPs that alter splicing or alternative splicing (AS), such as by changing the sequence-specific binding affinity of splicing factors to the pre-mRNA. We applied two methods in search for sQTLs by using RNA-seq data from control and lead-exposed w1118 Drosophila heads. First, we used the fraction of reads in a gene that falls in each exon as the phenotype. Second, we directly compared the transcript counts among the various splicing isoforms as the phenotype. Among the 1,236 potential Pb-responsive sQTLs (p < 0.0001, FDR < 0.39), mostly cis-sQTLs, one of the most distinct genes is Dscam1 (Down Syndrome Cell Adhesion Molecule), which has over 30,000 potential alternative splicing isoforms. We have also identified a candidate Pb-responsive trans-sQTL hotspot that appears to regulate 129 genes that are enriched in the “cation channel” gene ontology category, suggesting a model in which alternative splicing of these channels might lead to an increase in the elimination of Pb2+ from the neurons encoding these channels. To our knowledge, this is the first paper that uses sQTL analyses to understand the neurotoxicology of an environmental toxin in any organism, and the first reported discovery of a candidate trans-sQTL hotspot.

Lead Modulates trans- and cis-Expression Quantitative Trait Loci (eQTLs) in Drosophila melanogaster Heads

“…This enables the detection of structural variations such as alternative splicing and novel transcripts. And we successfully used this set of RNA-seq data to search for splicing QTLs (sQTLs)—QTLs that were associated with splicing events, both cis -sQTLs and trans -sQTLs (Ruden et al, 2017 ). Third, the abundant genotype information in RNA-seq data, which includes 11,768 underlying parental haplotype structures, makes it more likely to pinpoint the precise eQTL locus, while the previous microarray eQTL analysis only contain 92 genomic markers, each of which was at least 5 cM wide (Ruden et al, 2009 ).…”

Environmental influences on RNA processing: Biochemical, molecular and genetic regulators of cellular response

“…Splicing QTLs (sQTLs) have been identified in humans and in model organisms (Li et al, 2016; Hasin-Brumshtein et al, 2016; Ongen & Dermitzakis, 2015; Gutierrez-Arcelus et al, 2015; Qu, Gurdziel, Pique-Regi, & Ruden, 2017; Saha et al, 2017). Analysis of alternative splicing in LCLs, fibroblasts and T-cells from 204 individuals showed that most splicing events are shared across cell types (up to 80%) and identified genetic variants correlated with inter-individual variation in splicing (Gutierrez-Arcelus et al, 2015).…”

“…The functional relevance of these sQTLs is strengthened by an enrichment for signatures of positive selection among these sites and high overlap with SNPs associated with autoimmune and neurodegenerative diseases as assessed by genome-wide association studies (GWAS). Another study identified over 100 cis-sQTLs in response to lead exposure in Drosophila melanogaster heads (Qu et al, 2017). The study also identified a potential trans-sQTL hotspot that regulated a cluster of transcripts in response to lead, indicating that trans-acting polymorphisms may have large impacts on isoform composition in different environmental contexts.…”

Genome-Wide Identification of Splicing Quantitative Trait Loci (sQTLs) in Diverse Ecotypes of Arabidopsis thaliana

“…Genetic variants, such as single nucleotide polymorphisms (SNPs), can substantially regulate the expression of transcript isoforms by modulating splice sites, which can impact phenotypic diversity and susceptibility to diseases in humans (Singh and Cooper, 2012; Takata et al, 2017). Recent studies showed that 22% of SNPs that are associated with different human diseases affect splicing (Qu et al, 2017; Park et al, 2018). The advances in RNA-Seq and genotyping have augmented the opportunities to monitor genetic variants and quantify transcriptomic features that allow us to understand the genetic landscapes of AS (Smith et al, 2018).…”

“…To illuminate the role of genetic variations on AS in a large collection of highly diverse Arabidopsis lines, we sought to map sQTLs influencing AS. sQTLs spread across the genome can either act in cis - to disrupt the splicing of a proximal pre-mRNA by modulating splicing factors binding affinity to the pre-mRNA or in trans by regulating the splicing of distal pre-mRNA through altered expression of splicing regulators (Yoo et al, 2016; Qu et al, 2017).…”