Transcriptome-wide analysis of primary human endothelial cell responses to 1 hour of protein translation inhibition identify nonsense mediated decay targets and a non-codingSLC11A2exon as an acute biomarker

Abstract

ABSTRACTNonsense mediated decay (NMD) lowers the cellular concentration of spliced RNAs harboring premature termination codons (PTC), and inhibition has been proposed as a potential therapeutic method. Conversely, NMD plays regulatory roles throughout the eukaryotic kingdom, including when protein translation is inhibited acutely as part of the integrated stress response. To define tools for endothelial evaluations of therapeutic NMD inhibition, and quantification of subtle cellular stress states, natural endothelial-expressed targets were examined via whole transcriptome RNA sequencing of primary human microvascular endothelial cells (HMECs) treated for 1h with cycloheximide, a protein translation and NMD inhibitor. Genes differentially expressed after 1h cycloheximide overlapped with genes differentially expressed many days after NMD-specific knockdown in other cell types. For endothelial cells, customized novel scripts used 255,500 exons in media-treated HMEC and 261,725 exons in cycloheximide-treated HMEC to predict 1h cycloheximide-stabilized exons. RT-PCR and RNASeq validations in other endothelial cells highlighted exon 3B of the iron transporterSLC11A2(also known asNRAMP2/DMT1) as a novel exon in a transcript most consistently stabilized. Exact junctional alignments toSLC11A2exon 3B were confirmed in blood outgrowth endothelial cells (BOECs) from 3 donors at mean 5.9% (standard deviation 2.0%) of adjacent constitutive exon expression, increasing 3.7-fold following 1h treatment with cycloheximide. Relevance beyond endothelial cells is supported bySLC11A2’swide expression profiles, genome-wide associations with microcytic anemia, biomarker status for poor prognosis ovarian cancer, and exon 3B sequence in RefSeq non-coding transcript NR_183176.1. The studies contribute understanding to functions affected acutely by NMD/translation inhibition and provide a stimulus for further studies in experimental, stress, and therapeutic settings.