Inflammation Drives Alternative First Exon usage to Regulate Immune Genes including a Novel Iron Regulated Isoform of Aim2

Abstract

<jats:title>Abstract</jats:title><jats:p>Determining the layers of gene regulation within the innate immune response is critical to our understanding of the cellular responses to infection and dysregulation in disease. We identified a conserved mechanism of gene regulation in human and mouse via changes in alternative first exon (AFE) usage following inflammation, resulting in changes to isoform usage. Of these AFE events, we identified 50 unannotated transcription start sites (TSS) in mice using Oxford Nanopore native RNA sequencing, one of which is the cytosolic receptor for dsDNA and known inflammatory inducible gene, <jats:italic>Aim2</jats:italic>. We show that this unannotated AFE isoform of <jats:italic>Aim2</jats:italic> is the predominant isoform transcribed during inflammation and contains an iron-responsive element in its 5′UTR enabling mRNA translation to be regulated by iron levels. This work highlights the importance of examining alternative isoform changes and translational regulation in the innate immune response and uncovers novel regulatory mechanisms of <jats:italic>Aim2</jats:italic>.</jats:p><jats:sec><jats:title>Summary Sentence</jats:title><jats:p>Alternative first exon usage was the major splicing event observed in macrophages during inflammation, which resulted in the elucidation of a novel isoform and iron mediated regulatory mechanism of the protein coding gene, <jats:italic>Aim2</jats:italic>.</jats:p></jats:sec>

Generation of an isoform-level transcriptome atlas of macrophage activation

“…The analysis of the transcriptome we generated shows that differential isoform expression between conditions exists but is limited and most often associated with the differential usage of transcription start sites (TSSs) which is similar to observations we have previously made in mouse and human macrophages (9). This shows that the splicing of genes itself is very similar between the conditions we investigated.…”

Isoform-level transcriptome Atlas of Macrophage Activation

“…The analysis of the transcriptome we generated shows that differential isoform expression between conditions exists but is limited and most often associated with the differential usage of transcription start sites (TSSs) which is similar to observations we have previously made in mouse and human macrophages (Robinson et al, 2020) . This shows that the splicing of genes itself is very similar between the conditions we investigated.…”

“…To build on our previous work (Byrne et al, 2017;Robinson et al, 2020) and further push the limits of long-read technology to provide a resource for the innate immune research community, we set out to generate an isoform-level transcriptome atlas of macrophage activation by determining 1) what isoform of a given gene is expressed, 2) at what level, and 3) how isoform and gene expression change following Toll-like Receptor (TLR) activation.…”

Nanopore direct RNA sequencing detects differential expression between human cell populations

“…Previous studies have demonstrated dRNA sequencing is a highly accurate method for quantifying expression of spike-in controls (Garalde et al 2018;Sessegolo et al 2019) and could identify differential gene expression in yeast (Jenjaroenpun et al 2018). DGE and/or DTE have recently been reported in C.elegans, Arabidopsis and mammalian cells with dRNA (Zhang et al 2020;Li et al 2020;Robinson et al 2020). However, these have almost exclusively been identified from fold changes alone or using very lenient statistics, leaving the capability of dRNA to identify differential expression in a robust manner uncertain.…”