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Updated: Oct 24, 2025
Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA
Published on: November 14, 2017
Nanopore sequencing reveals endogenous NMD-targeted isoforms in human cells
Evangelos D Karousis1, Foivos Gypas2, Mihaela Zavolan3
1Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Freiestrasse 3, 3012, Bern, Switzerland.
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.
Insights
Nonsense-mediated mRNA decay (NMD) targets faulty transcripts and regulates gene expression. Long-read sequencing identified novel NMD targets, revealing its role in managing spurious splicing events and gene regulation.
Area of Science:
- Molecular Biology
- Genomics
- Transcriptomics
Background:
- Nonsense-mediated mRNA decay (NMD) is a critical eukaryotic pathway.
- NMD degrades mRNAs with premature termination codons and regulates full-length protein expression.
- Identifying NMD targets, especially from short-read sequencing data, is challenging.
Purpose of the Study:
- To identify and analyze endogenous NMD targets in human cells.
- To investigate the role of NMD in regulating gene expression and transcript stability.
- To characterize NMD-sensitive isoforms and their biogenesis.
Main Methods:
- Application of cDNA Nanopore sequencing and short-read sequencing.
- Analysis of human cells with varying NMD factor expression levels.
- Isoform-aware analysis to detect changes in splicing and expression.
Main Results:
- Detection of highly unstable, full-length NMD substrates that appear or increase upon NMD inhibition.
- Identification of numerous novel NMD-targeted isoforms, predominantly arising from alternative exon usage.
- Discovery that NMD-sensitive mRNAs possess more 3′ UTR exons, and 3′ UTR length alone does not dictate sensitivity.
Conclusions:
- Long-read sequencing facilitates the discovery of novel NMD-sensitive mRNAs.
- NMD plays a role in regulating gene expression through alternative splicing and clearing spurious transcripts.
- The study provides a valuable resource for human NMD transcript targets in future research.
