NCM 2021: Nanopore sequencing reveals endogenous NMD-targeted isoforms in human cells

Evangelos discussed how his lab used nanopore sequencing to assess mRNAs that are targeted by nonsense-mediated mRNA decay (NMD) in human cells. NMD is a translation-dependent process that leads to the degradation of targeted mRNAs. NMD targets aberrant mRNAs with premature termination codons (PTC), but it can also target endogenous mRNAs which encode full-length proteins. Certain factors are important in the degradation process and these factors can be experimentally depleted, leading to the accumulation of mRNAs, making it possible to identify the substrates of NMD. Evangelos ‘knocked down NMD factors’ in killer cells to study NMD activity. The NMD-sensitive mRNAs that accumulated were detected by nanopore sequencing. The long reads were used as a reference to map short sequencing reads and quantify splicing isoforms that were absent from previous annotations. NMD factors were isolated from single and double knockdown conditions, and cDNA libraries were prepared for direct cDNA sequencing on a GridION. The long-read sequences revealed many previously unannotated exons. The most common event to produce NMD-sensitive transcripts was the introduction of alternative exons, ‘these arise either by an exon being included or excluded, compared to the NMD-insensitive isoform.’ Evangelos explained how long-read sequencing enables the identification of novel RNA isoforms using UTP11 as an example. The most known NMD-inducing feature is the presence of an exon junction downstream of the termination codon. This was confirmed in the study, and with this in mind, Evangelos addressed a long-standing question: ‘whether NMD also targets endogenous mRNAs with long 3’UTRs independently of exon junctions’ and suggested that ‘NMD targets mRNAs with exon junctions in the 3’-UTR independently of its length’. NMD employs two methods in saving the transcriptome; the first is the clearance of transcripts resulting from aberrant splicing and the second is the degradation of transcripts with highly conserved alternative exons.