Journal Article

Nanopore sequencing reveals endogenous NMD-targeted isoforms in human cells

Zavolan, Mihaela, Karousis, Evangelos D., Mühlemann, Oliver, Gypas, Foivos

2021

Overview

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 apply 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 identifies, most derive from alternative exon usage. The isoform-aware analysis reveals 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 enables the identification of many novel NMD-sensitive mRNAs and reveals 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.

• Publisher: BioMed Central,Springer Nature B.V,BMC
• Subject: 3' Untranslated regions / Alternative splicing / Animal Genetics and Genomics / biogenesis / Bioinformatics / Biomedical and Life Sciences / Cancer / Carrier Proteins - genetics / cDNA sequencing / Codon, Nonsense / Codons / Evolutionary Biology / Exons / Gene expression / gene expression regulation / Genomics / HeLa Cells / Human Genetics / Humans / Isoforms / Kinases / Life Sciences / Long Read Sequencing / Microbial Genetics and Genomics / mRNA degradation / mRNA turnover / Nanopore sequencing / Nanopore Sequencing - methods / nanopores / NMD / Nonsense Mediated mRNA Decay / Plant Genetics and Genomics / Protein Isoforms - genetics / Proteins / RNA Splicing / RNA Stability / RNA, Messenger - genetics / stop codon / Telomerase - genetics / Transcription / Transcriptome / Transcriptomes / transcriptomics