Deep sequencing of pre-translational mRNPs reveals hidden flux through evolutionarily conserved AS-NMD pathways
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ABSTRACT:
PROVIDER: PRJNA631548 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11747000_1.fastq.gz | Fastqsanger.gz | |||
| SRR11747000_2.fastq.gz | Fastqsanger.gz | |||
| SRR11747001_1.fastq.gz | Fastqsanger.gz | |||
| SRR11747001_2.fastq.gz | Fastqsanger.gz | |||
| SRR11746990_1.fastq.gz | Fastqsanger.gz |
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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.
2021-02-19 | GSE150228 | 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 | PXD027479 | Pride
Project description:BackgroundAlternative splicing, which generates multiple mRNA isoforms from single genes, is crucial for the regulation of eukaryotic gene expression. The flux through competing splicing 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.ResultsRNA immunoprecipitation in tandem (RIPiT) of exon junction complex 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 compare exon junction complex 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 RIPiT-Seq also definitively demonstrates that the splicing machinery itself has no ability to detect reading frame. We identify 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.ConclusionsDeep 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 nonsense-mediated decay-linked alternative splicing pathway predominates. Exon junction complex RIPiT-Seq also revealed numerous conserved but previously unannotated AS-NMD events.
| S-EPMC8091538 | biostudies-literature
Project description:Nonsense mediated decay (NMD) is a translation termination coupled quality control system. NMD identifies and degrades aberrant mRNAs containing premature termination codons, thereby preventing the accumulation of detrimental truncated proteins. NMD also controls the expression of several normal mRNAs and non-coding transcripts. RNA-seq assays were conducted to identify the NMD regulated genes in Arabidopsis. However, NMD targets have not been studied in other plants. To identify the conserved NMD targets, we wanted to identify the NMD regulated genes in Nicotiana benthamiana model species. Virus induced gene silencing was used to inactivate UPF1 NMD key factor in N. benthamiana, and then comparative RNA-seq experiment was carried out using UPF1-silenced and control–silenced plants. We found that in N. benthamiana significantly more genes are controlled by NMD than in Arabidopsis and that, surprisingly few conserved NMD targets can be recognized. These results suggest that while the mechanism of NMD is highly conserved, the set of NMD controlled genes can be quickly changed in plants.
2019-07-01 | GSE109130 | GEO
Project description:Nonsense-mediated mRNA decay (NMD) is a major translation-dependent RNA degradation pathway required for embryo development and telomere maintenance. Core NMD factors Upf1, Upf2 and Upf3 are conserved from yeast to mammals but a model of the NMD machinery compatible with all eukaryotes is not yet available. We performed the first large-scale quantitative characterization of yeast NMD complexes through affinity purification and mass-spectrometry with 7 different NMD-related factors, with or without Rnase, in strains deleted or not for NMD genes. This extensive characterization of NMD complexes identified two distinct complexes associated with Upf1: Detector (Upf1/2/3) and Effector. Effector contained, in addition to Upf1, the mRNA decapping enzyme and two potential equivalents of mammalian Smg6/5/7: Nmd4 and Ebs1. Like the Smg proteins, Nmd4 and Ebs1 were required for efficient NMD. Our results suggest that the core eukaryotic NMD machinery is conserved across species and operates through successive Upf1-bound Detector and Effector complexes.
2018-03-12 | PXD007159 | Pride
Project description:Nonsense-mediated decay (NMD) is a pathway that degrades messenger RNAs containing premature termination codons. Here, a genome-wide screen for NMD factors uncovered an unexpected mechanism that broadly governs 3ʹ untranslated region (UTR)-directed regulation. The screen revealed that NMD requires lysosomal acidification, which allows transferrin-mediated iron uptake, which in turn is necessary for iron-sulfur (Fe-S) cluster biogenesis. This pathway converges on the Fe-S cluster-containing ribosome recycling factor ABCE1, whose impaired function results in the movement of ribosomes into 3ʹ UTRs where they displace exon junction complexes, thereby abrogating NMD. Importantly, these effects extend beyond NMD substrates, with ABCE1 activity required to maintain the accessibility of 3ʹ UTRs to diverse regulators, including microRNAs and RNA binding proteins. Due to the sensitivity of the Fe-S cluster of ABCE1 to iron availability and reactive oxygen species, these findings reveal an unanticipated vulnerability of 3ʹ UTR-directed regulation to lysosomal dysfunction, iron deficiency, and oxidative stress.
2020-07-14 | GSE144162 | GEO
Project description:We report a high resolution catalouge of NMD substrates using RNA-Seq. We discovered several hundred new substrates for NMD. Using published ribosome footprint profiling data, we measured ribosome densities of normal-looking NMD substrates and non-NMD substrates. NMD substrates exhibited a striking difference in normalized ribosome occupancy in wild-type and UPF1 cells. We also found that normal looking NMD substrates have higher ratio of out of frame reads, lower codon optimalites and a higher propensity to have long stretches of non-optimal codons.
2016-12-02 | GSE86428 | GEO
Project description:The nonsense-mediated mRNA decay pathway (NMD) is a quality control mechanism that detects and degrades mRNAs that contain premature translation termination codons (PTCs), thus preventing the production of potentially harmful truncated proteins. NMD has been studied in a number of organisms including humans, yeast, Drosophila, C. elegans and recently Arabidopsis. Recently the NMD process has been shown to have a wider significance in regulating the levels of mRNA expression for a wide range of genes in yeast and Drosophila. Several genes have been found to be required for NMD in a variety of organisms, including UPF1 and UPF3. We have identified and characterised a number of alleles of UPF1 and UPF3 in Arabidopsis that are defective in NMD. By doing a transcriptomic analysis of two NMD mutants, upf1-1 and upf3-1, and wild type plants we aim to identify the subset of genes that are co-regulated by UPF1 and UPF3 and, therefore, by NMD in Arabidopsis. RNA will be extracted from 17-day-old mutant and wild type seedlings grown at 22-24 C under constant light.
2010-07-09 | GSE19253 | GEO
Project description:Nonsense-mediated mRNA decay (NMD) is a conserved mRNA quality control mechanism that identifies and destroys aberrant mRNAs that contain premature termination codons (PTCs). The NMD core comprises seven proteins, from SMG1 to SMG7. Arabidopsis has orthologues of most of these proteins. Studies on yeast, Drosophila, Humans and Arabidopsis reveal that NMD not only functions to target PTC-containing mRNAs but also acts as a global regulator of gene expression. It has also been reported that the NMD pathway branches with some target genes being dependent on specific NMD factors. In order to determine the extent to which different NMD factors co-regulate or independently regulate Arabidopsis genes, transcriptome analysis of smg7b-1 mutants will be carried out and added to existent data of upf1, upf3 and smg5 NMD mutants for comparison. RNA will be extracted from 17 day-old mutant and wild type seedlings grown at 22-24 C under constant light.
2011-10-07 | GSE32672 | GEO
Project description:CRISPR screen for NMD regulators
| PRJNA602918 | ENA