Nanopore sequencing reveals endogenous NMD-targeted isoforms in human cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB44747 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR5880581.fastq.gz | Fastqsanger.gz | |||
| ERR5880582.fastq.gz | Fastqsanger.gz | |||
| ERR5880583.fastq.gz | Fastqsanger.gz | |||
| ERR5880584.fastq.gz | Fastqsanger.gz | |||
| ERR5880585.fastq.gz | Fastqsanger.gz |
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Project description: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. 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 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, but a main function is most likely to rid the transcriptome of isoforms resulting from spurious splicing events. 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.
2021-07-20 | E-MTAB-10452 | biostudies-arrayexpress
Project description:BackgroundNonsense-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.ResultsTo 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.ConclusionsLong-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.
| S-EPMC8361881 | biostudies-literature
Project description:Analysis of miRNA-targeted cellular NMD substrates in HeLa cell. The hypothesis tested in the present study was that endogenous NMD substrates containing long 3' untranslated region may targeted for miRNA. Results provide important information expanding the roles of miRISC in the posttranscriptional regulation of gene expression: a new cross-talk between miRNA-mediated gene silencing and NMD. ABSTRACT: Imperfect base-pairing between microRNA (miRNA) and the 3’-untranslated region (3’UTR) of target mRNA triggers translational repression of the target mRNA. Here, we provide evidence that human Argonaute 2 (Ago2) targets cap-binding protein (CBP)80/20- and exon junction complex (EJC)-bound mRNAs and inhibits nonsense-mediated mRNA decay (NMD), which is tightly restricted to CBP80/20-bound mRNAs. Furthermore, microarray analyses reveal that a subset of cellular transcripts, which are expected to be targeted for NMD, is stabilized by miRNA-mediated gene silencing. The regulation of NMD by miRNAs will shed light on a new post-transcriptional regulation mechanism of gene expression in mammalian cells
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Project description:Analysis of miRNA-targeted cellular NMD substrates in HeLa cell. The hypothesis tested in the present study was that endogenous NMD substrates containing long 3' untranslated region may targeted for miRNA. Results provide important information expanding the roles of miRISC in the posttranscriptional regulation of gene expression: a new cross-talk between miRNA-mediated gene silencing and NMD. ABSTRACT: Imperfect base-pairing between microRNA (miRNA) and the 3â-untranslated region (3âUTR) of target mRNA triggers translational repression of the target mRNA. Here, we provide evidence that human Argonaute 2 (Ago2) targets cap-binding protein (CBP)80/20- and exon junction complex (EJC)-bound mRNAs and inhibits nonsense-mediated mRNA decay (NMD), which is tightly restricted to CBP80/20-bound mRNAs. Furthermore, microarray analyses reveal that a subset of cellular transcripts, which are expected to be targeted for NMD, is stabilized by miRNA-mediated gene silencing. The regulation of NMD by miRNAs will shed light on a new post-transcriptional regulation mechanism of gene expression in mammalian cells Total RNA obtained from HeLa cells with downregulation of Ago2 or Ago2/UPF1 by siRNA. The up- or down-regulated transcripts were compared to control siRNA treated HeLa cell RNA extract. Significant transcripts were confirmed by replication.
2010-04-09 | E-GEOD-16170 | biostudies-arrayexpress
Project description:Background: Alternative splicing (AS), which generates multiple mRNA isoforms from single genes, is crucial for the regulation of eukaryotic gene expression. The flux through competing AS pathways cannot be determined by traditional RNA-Seq, however, because different mRNA isoforms can have widely differing decay rates. Indeed, some mRNA isoforms with extremely short half-lives, such as those subject to translation-dependent nonsense-mediated decay (AS-NMD), may be completely overlooked in even the most extensive RNA-Seq analyses. Results: RNA immunoprecipitation in tandem (RIPiT) of exon junction complex (EJC) components allows for purification of post-splicing mRNA-protein particles (mRNPs) not yet subject to translation (pre-translational mRNPs) and, therefore, translation-dependent mRNA decay. Here we compared EJC RIPiT-Seq to whole cell RNA-Seq data from HEK293 cells. Consistent with expectation, the flux through known AS-NMD pathways is substantially higher than that captured by RNA-Seq. Our EJC RIPiT-Seq also definitively demonstrates that the splicing machinery itself has no ability to detect reading frame. We identified thousands of previously unannotated splicing events; while many can be attributed to “splicing noise”, others are evolutionarily-conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis. Conclusions: Deep sequencing of RNAs in post-splicing, pre-translational mRNPs provides a means to identify and quantify splicing events without the confounding influence of differential mRNA decay. For many known AS-NMD targets, the NMD-linked AS pathway predominates. EJC RIPiT-Seq also enabled identification of numerous conserved but previously unknown AS-NMD events.
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Project description:Two core factors of the NMD machinery in D. melanogaster, Upf1 and Upf2, were knocked down using RNAi, as described in Rehwinkel et al. (2005) RNA, PMID: 16199763. Each of the two knockdowns were compared to mock RNAi knockdowns as described in Blanchette et al. (2005) Genes Dev, PMID: 15937219 in a dual channel experiment, using a custom splice-junction microarray design, see Blanchette et al. (2005), PMID: 15937219. The aim of the experiment was to identify which isoforms of alternatively spliced genes were affected by NMD knockdown and thereby gain insight into the NMD mechanism in Drosophila. The samples were hybridized in a dual channel setup, to custom designed Splice-Junction arrays manufactured by Agilent.
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