The genetic basis for individual differences in mRNA splicing and APOBEC1 editing activity in murine macrophages

Abstract

Alternative splicing and mRNA editing are known to contribute to transcriptome diversity. Although alternative splicing is pervasive and contributes to a variety of pathologies, including cancer, the genetic context for individual differences in isoform usage is still evolving. Similarly, although mRNA editing is ubiquitous and associated with important biological processes such as intracellular viral replication and cancer development, individual variations in mRNA editing and the genetic transmissibility of mRNA editing are equivocal. Here, we have used linkage analysis to show that both mRNA editing and alternative splicing are regulated by the macrophage genetic background and environmental cues. We show that distinct loci, potentially harboring variable splice factors, regulate the splicing of multiple transcripts. Additionally, we show that individual genetic variability at the Apobec1 locus results in differential rates of C-to-U(T) editing in murine macrophages; with mouse strains expressing mostly a truncated alternative transcript isoform of Apobec1 exhibiting lower rates of editing. As a proof of concept, we have used linkage analysis to identify 36 high-confidence novel edited sites. These results provide a novel and complementary method that can be used to identify C-to-U editing sites in individuals segregating at specific loci and show that, beyond DNA sequence and structural changes, differential isoform usage and mRNA editing can contribute to intra-species genomic and phenotypic diversity. [Supplemental material is available for this article.]Splicing is an obligatory step in the processing of both coding and noncoding RNA precursors (pre-RNA) to mature RNA, and is executed by a ribonucleoprotein megaparticle known as the spliceosome. Even though the sequential assembly of the spliceosome (Kornblihtt et al. 2013) can occur around any splice site that consists of consensus sequences recognized by the spliceosomal components, splice sites that conform better to a consensus sequence are strongly recognized and favored by the spliceosome complex. Besides the consensus sequences, the selection of optimal splice sites is regulated, in part, by a minimal set of conserved cis-acting GU and AG sequences at the 59 and 39 splice sites, respectively , and sequence elements known as intronic/exonic splicing enhancers and silencers

Genetic Architectures of Quantitative Variation in RNA Editing Pathways

“…These findings are consistent with a recent report of editing variation in the AXB/BXA recombinant inbred panel (Hassan et al 2014). Of the remaining C-to-U editing sites, 11 show local genetic associations that map to the region of the edited gene.…”

“…Another study found evidence of genetic variation for two (of 7389) A-to-I editing sites (Daneck et al 2012). Still another study found an association between RNA editing rates and genetic variation of Apobec1 (Hassan et al 2014). The broader impact of genetic variation on RNA editing in humans and mice remains unclear.…”

Genome-wide analysis of differential RNA editing in epilepsy

“…S3). Additionally, we identified 10 transcripts with 68 C-to-U sites DRE between epileptic and control mouse hippocampus, which also included four C-to-U sites which had been previously reported as RNA-editing events in macrophages (Supplemental Table S2; Hassan et al 2014).…”

Incorporating alternative splicing and mRNA editing into the genetic analysis of complex traits

“…In fact, it can be argued that the success of genetical genomics and its incorporation into the genetics of complex traits is largely due to the heritability of mRNA abundance, not to mention the simplicity of performing large-scale transcriptional analysis. Recently, we integrated isoform usage and linkage analyses in a genetically divergent set of recombinant inbred (RI) mice, and showed that isoform usage varies with both the macrophage genetic background and physiological state [9]. For instance, we observed that the C-type lectin domain family 7 member a (Clec7a or Dectin-1) gene, from which an alternative transcript that lacks the 4th exon can be produced [32], is differentially spliced in bone marrow-derived macrophages (BMDM) obtained from the classical laboratory inbred A/J (AJ) and C57BL/6J (B6) mice.…”

“…A major drawback to genetical genomics is that it presupposes that mRNA abundance represents the entirety of transcriptome complexity. Emerging empirical evidence indicates that several factors, such as alternative splicing (AS) and mRNA editing, not only contribute to transcriptome diversity but also are variable among individuals [9]. In fact, often protein abundance does not mirror the steady state transcript levels [10,11].…”