Identification of translation events that drive nonsense-mediated mRNA decay reveals functional roles for alternative RNA isoforms
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE308938 | GEO | 2026/09/16
REPOSITORIES: GEO
Similar Datasets
Project description:Gene expression is a product of RNA synthesis and degradation kinetics, which can vary among transcript isoforms of the same gene. Some isoforms contain premature termination codons (PTCs) and are degraded via nonsense mediated mRNA decay (NMD), while others lack these features and are more stable. However, probing the stability of individual isoforms remains challenging. Methods like nucleotide-recoding RNA-seq (NR-seq) enable insights into RNA kinetics, but existing bioinformatic tools are unable to provide robust, isoform-specific degradation rate constant estimates. We present a strategy to infer isoform-level kinetics from short-read NR-seq data, implemented in the EZbakR-suite, which allows us to identify high confidence NMD targets. We also show how custom annotations improve these analyses. We uncovered unexpected stability variability in transcripts with conserved PTC-containing exons and found that other decay pathways can supersede NMD. Our findings provide mechanistic insights into established NMD efficiency correlates and highlight transcript features promoting efficient decay.
2026-02-10 | GSE289489 | GEO
Project description:Eukaryotic RNAs with premature termination codons (PTCs) are eliminated by nonsense-mediated decay (NMD). While human nonsense RNA degradation can be initiated either by an endonucleolytic cleavage event near the PTC or through decapping, the individual contribution of these activities on endogenous substrates has remained unresolved. Here we unambiguously establish that SMG6-catalyzed endonucleolysis is the primary initiating step in human nonsense RNA decay. We also show that both protein-coding and ‘non-coding’ genes hosting snoRNAs in their introns produce considerable amounts of NMD-sensitive splice variants, suggesting that these RNAs are merely by-products of a primary snoRNA production process. Finally, genes encoding multiple snoRNAs generally yield elevated numbers of alternative transcript isoforms, enabling the differential expression of individual snoRNAs. These findings demonstrate a hitherto unappreciated potential for decoupling of the individual expression levels of functional exon- and intron-encoded species from such composite genes. HEK293 Flp-In T-Rex cells were subjected to siRNA-mediated depletion of XRN1 and co-depletion of XRN1 with either SMG6 or UPF1. All the treated samples together with the controls were subjected to both RNA-seq and 5'-end-seq. RNA-seq was used for detecting NMD isoforms and their expression levels. 5'-end-seq was used for finding NMD decay intermediates (decapped and endocleaved molecules). CAGE was used to distinguish decapped from endocleaved RNA fragments.
2014-11-18 | E-GEOD-57433 | biostudies-arrayexpress
Project description:Elevated temperature enhances the frequency of alternative RNA splicing (AS) in plants producing RNA isoforms, some of which are productive, likely expanding the repertoire of stress responses, while other isoforms are nonproductive or possibly toxic. This raises the question whether plants can recognize and degrade nonproductive RNA isoforms by nonsense-mediated decay (NMD). In this study Arabidopsis seedlings were subjected to heat stress and RNA isoforms were identified that were upregulated by heat treatment but spared degradation in NMD mutants or in seedlings in which translation was blocked by cycloheximide. The differentially expressed isoforms (DEIs) produced in response to heat stress and that might be relevant to NMD were grouped according to expression patterns. Group 1 DEIs were rapidly heat-induced, then declined during futher heat treatment, but the transcripts were spared from degradation when NMD was compromised. Group 2 DEIs were not upregulated in response to heat; however, their transcript levels declined during heat treatment, but were spared when NMD was blocked. Group 3 DEIs were constitutively expressed although at higher levels when NMD was blocked. While the common targets of NMD are RNA transcripts with premature termination codons (PTCs), other seemingly normal transcripts, such as those with long 3’-untranslated regions (3’-UTRs) are probablistics NMD targets. Long 3’-UTRs from some Arabidopsisi genes such as WRKY70, ACS11, YUCC5, OSM, and SARD1, were confirmed to be NMD targets using a modified Dual-Luciferase (LUC) 3’-UTR test system in protoplasts. Following heat stress, upstream frameshift (upf) mutants tended to spare from degradation NMD targets, such as transcripts with PTCs. Despite their penchant for sparing transcripts from degradation, upf mutants were more sensitive to stress, which demonstrates the importance of NMD to resist abiotic stresses, such as heat stress.
2026-07-08 | GSE232084 | GEO
Project description:Translation of messenger RNAs (mRNAs) with premature translation termination codons produces truncated proteins with potentially deleterious effects. This is prevented by nonsense-mediated mRNA decay (NMD) of these mRNAs. NMD is triggered by ribosomes terminating upstream of a splice site marked by an exon-junction complex (EJC), but also acts on many mRNAs lacking a splice junction after their termination codon. We developed a genome-wide CRISPR flow cytometry screen to identify regulators of mRNAs with premature termination codons in K562 cells. This screen recovered essentially all core NMD factors and suggested a role for EJC factors in degradation of PTCs without downstream splicing. Among the strongest hits were the translational repressors GIGYF2 and EIF4E2. GIGYF2 and EIF4E2 mediate translational repression but not mRNA decay of a subset of NMD targets and interact with NMD factors genetically and physically. Our results suggest a model wherein recognition of a stop codon as premature can lead to its translational repression through GIGYF2 and EIF4E2.
2021-10-12 | PXD027487 | Pride
Project description:Translation of messenger RNAs (mRNAs) with premature translation termination codons produces truncated proteins with potentially deleterious effects. This is prevented by nonsense-mediated mRNA decay (NMD) of these mRNAs. NMD is triggered by ribosomes terminating upstream of a splice site marked by an exon-junction complex (EJC), but also acts on many mRNAs lacking a splice junction after their termination codon. We developed a genome-wide CRISPR flow cytometry screen to identify regulators of mRNAs with premature termination codons in K562 cells. This screen recovered essentially all core NMD factors and suggested a role for EJC factors in degradation of PTCs without downstream splicing. Among the strongest hits were the translational repressors GIGYF2 and EIF4E2. GIGYF2 and EIF4E2 mediate translational repression but not mRNA decay of a subset of NMD targets and interact with NMD factors genetically and physically. Our results suggest a model wherein recognition of a stop codon as premature can lead to its translational repression through GIGYF2 and EIF4E2.
2021-10-12 | PXD027479 | Pride
Project description:Eukaryotic RNAs with premature termination codons (PTCs) are eliminated by nonsense-mediated decay (NMD). While human nonsense RNA degradation can be initiated either by an endonucleolytic cleavage event near the PTC or through decapping, the individual contribution of these activities on endogenous substrates has remained unresolved. Here we unambiguously establish that SMG6-catalyzed endonucleolysis is the primary initiating step in human nonsense RNA decay. We also show that both protein-coding and ‘non-coding’ genes hosting snoRNAs in their introns produce considerable amounts of NMD-sensitive splice variants, suggesting that these RNAs are merely by-products of a primary snoRNA production process. Finally, genes encoding multiple snoRNAs generally yield elevated numbers of alternative transcript isoforms, enabling the differential expression of individual snoRNAs. These findings demonstrate a hitherto unappreciated potential for decoupling of the individual expression levels of functional exon- and intron-encoded species from such composite genes. ------------------------------------------------------------------------------------- This article was corrected in http://genesdev.cshlp.org/content/30/9/1128.short Completely mapped 5'p-end sequencing data from the corrigendum have been updated in corresponding samples.
2014-11-18 | GSE57433 | GEO
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:All eukaryotes studied to date have the capacity to detect and degrade mRNAs harboring premature translation termination codons (PTCs) in a process called nonsense-mediated mRNA decay (NMD) (reviewed in Wagner E & Lykke-Andersen J, 2002). This surveillance system allows the cell to prevent the expression of potentially harmful truncated proteins. trans-acting factors required for NMD in Drosophila are the proteins UPF1, UPF2, UPF3, SMG-1, SMG-5 and SMG-6. To identify mRNAs naturally regulated by NMD, we analyzed expression profiles in Drosophila cells RNAi-depleted of known NMD factors using high-density oligonucleotide arrays.
2005-11-01 | E-MEXP-202 | biostudies-arrayexpress
Project description:A large number of genetic diseases are caused by various mutations in specific disease genes. A significant proportion (~15%) of these mutations are nonsense mutations that create a premature termination codon (PTC). Consequently, the Nonsense-mediated mRNA Decay (NMD) surveillance pathway degrades a large fraction of PTC-containing mRNA. Translation of the remaining un-degraded PTC-containing mRNA terminates at the PTC, leading to no full-length protein production and hence disease. Therefore, suppressing NMD and translation termination at PTCs becomes an attractive strategy for combating these diseases. This work presents a novel approach, namely targeted PTC pseudouridylation, to suppress nonsense mutations in human cells. By co-transfecting human cells with a PTC-reporter gene and a designer box H/ACA guide RNA gene targeting the PTC, we show that targeted pseudouridylation suppresses both NMD and translation termination at PTCs in the contexts of various disease gene sequences. Furthermore, targeted pseudouridylation exhibits a level of suppression comparable to that of antibiotic treatments, and the suppressive effect is long-lasting in the cell. When targeted pseudouridylation is combined with the antibiotic treatment, a much higher level of suppression is observed. Remarkably, by transfecting a disease model cell line (carrying a chromosomal PTC) with a designer guide RNA gene alone targeting the PTC, we also observe nonsense suppression, suggesting the high potential of this novel approach in treating PTC-associated diseases. Finally, one of the restored full-length proteins is further tested to be functional. Targeted pseudouridylation appears to be the first RNA-directed gene-specific therapeutic approach that suppresses NMD and concurrently promotes PTC read-through.
2022-12-31 | GSE221516 | GEO
Project description:Long Undecoded Transcript Isoforms (LUTIs) represent a class of non-canonical mRNAs that downregulate gene expression through the combined act of transcriptional and translational repression. While single gene studies revealed some important aspects of LUTI-based repression, how these features impact gene regulation at a global scale is unknown. By using transcript leader and direct RNA sequencing, here we identify 74 LUTI candidates that are expressed specifically during meiotic prophase. Translational repression of these candidates is ubiquitous and dependent on upstream open reading frames. However, LUTI-based transcriptional repression is highly variable. In only 50% of the cases, LUTI transcription causes downregulation of the protein-coding transcript isoform. Higher LUTI expression, enrichment of histone 3 lysine 36 trimethylation, and changes in nucleosome position are the strongest predictors of LUTI-based transcriptional repression. We conclude that LUTIs downregulate gene expression in a manner that integrates translational repression, chromatin state changes, and the magnitude of LUTI expression.
2020-11-01 | GSE140177 | GEO