PT  - JOURNAL ARTICLE
AU  - Evangelos D. Karousis
AU  - Foivos Gypas
AU  - Mihaela Zavolan
AU  - Oliver Mühlemann
TI  - Nanopore sequencing reveals endogenous NMD-targeted isoforms in human cells
AID  - 10.1101/2021.04.30.442116
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.04.30.442116
4099  - http://biorxiv.org/content/early/2021/07/01/2021.04.30.442116.short
4100  - http://biorxiv.org/content/early/2021/07/01/2021.04.30.442116.full
AB  - Background Nonsense-mediated mRNA decay (NMD) is a eukaryotic, translation-dependent degradation pathway that targets mRNAs with premature termination codons and also regulates the expression of some mRNAs that encode full-length proteins. Although many genes express NMD-sensitive transcripts, identifying them based on short-read sequencing data remains a challenge.Results To identify and analyze endogenous targets of NMD, we applied cDNA Nanopore sequencing and short-read sequencing to human cells with varying expression levels of NMD factors. Our approach detects full-length NMD substrates that are highly unstable and increase in levels or even only appear when NMD is inhibited. Among the many new NMD-targeted isoforms that our analysis identified, most derive from alternative exon usage. The isoform-aware analysis revealed many genes with significant changes in splicing but no significant changes in overall expression levels upon NMD knockdown. NMD-sensitive mRNAs have more exons in the 3΄UTR and, for those mRNAs with a termination codon in the last exon, the length of the 3΄UTR per se does not correlate with NMD sensitivity. Analysis of splicing signals reveals isoforms where NMD has been co-opted in the regulation of gene expression, though the main function of NMD seems to be ridding the transcriptome of isoforms resulting from spurious splicing events.Conclusions Long-read sequencing enabled the identification of many novel NMD-sensitive mRNAs and revealed both known and unexpected features concerning their biogenesis and their biological role. Our data provide a highly valuable resource of human NMD transcript targets for future genomic and transcriptomic applications.Competing Interest StatementThe authors have declared no competing interest.NMDNonsense-mediated mRNA decayAS-NMDAlternative splicing linked to NMDEJCExon junction complexKDKnockdowndKDDouble knockdownTCTermination codonPTCPremature termination codonPSIPercent spliced inORFOpen reading frameuORFUpstream open reading frame