NMD targets experience deadenylation during their maturation and endonucleolytic cleavage during their decay.

Premature stop codon-containing mRNAs can produce truncated and dominantly acting proteins that harm cells. Eukaryotic cells protect themselves by degrading such mRNAs via the Nonsense-Mediated mRNA Decay (NMD) pathway. The precise reactions by which cells attack NMD target mRNAs remain obscure, precluding a mechanistic understanding of NMD and hampering therapeutic efforts to control NMD. A key step in NMD is the decay of the mRNA, which is proposed to occur via several competing models including deadenylation, exonucleolytic decay, and/or endonucleolytic decay. We set out to clarify the relative contributions of these decay mechanisms to NMD, and to identify the role of key factors. Here, we modify and deploy single-molecule nanopore mRNA sequencing to capture full-length NMD targets and their degradation intermediates, and we obtain single-molecule measures of splicing isoform, cleavage state, and poly(A) tail length. We observe robust endonucleolytic cleavage of NMD targets in vivo that depends on the nuclease SMG-6 and we use the occurence of cleavages to identify several known NMD targets. We show that NMD target mRNAs experience deadenylation, but similar to the extent that normal mRNAs experience as they enter the translational pool. Furthermore, we show that a factor (SMG-5) that historically was ascribed a function in deadenylation, is in fact required for SMG-6-mediated cleavage. Our results support a model in which NMD factors act in concert to degrade NMD targets in animals via an endonucleolytic cleavage near the stop codon, and suggest that deadenylation is a normal part of mRNA (and NMD target) maturation rather than a facet unique to NMD. Our work clarifies the route by which NMD target mRNAs are attacked in an animal.

Poly(a) selection introduces bias and undue noise in direct RNA-sequencing.

BACKGROUND: Genome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA, with the most popular enrichment method being poly(A) selection. In many applications it is well-known that poly(A) selection biases the view of the transcriptome by selecting for longer tailed mRNA species. RESULTS: Here, we show that poly(A) selection biases Oxford Nanopore direct RNA sequencing. As expected, poly(A) selection skews sequenced mRNAs toward longer poly(A) tail lengths. Interestingly, we identify a population of mRNAs (> 10% of genes' mRNAs) that are inconsistently captured by poly(A) selection due to highly variable poly(A) tails, and demonstrate this phenomenon in our hands and in published data. Importantly, we show poly(A) selection is dispensable for Oxford Nanopore's direct RNA-seq technique, and demonstrate successful library construction without poly(A) selection, with decreased input, and without loss of quality. CONCLUSIONS: Our work expands the utility of direct RNA-seq by validating the use of total RNA as input, and demonstrates important technical artifacts from poly(A) selection that inconsistently skew mRNA expression and poly(A) tail length measurements.