Global SLAM-Seq for accurate mRNA decay determination and identification of NMD targets
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ABSTRACT:
PROVIDER: PRJNA806863 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR18015213.fastq.gz | Fastqsanger.gz | |||
| SRR18015214.fastq.gz | Fastqsanger.gz | |||
| SRR18015215.fastq.gz | Fastqsanger.gz | |||
| SRR18015216.fastq.gz | Fastqsanger.gz | |||
| SRR18015217.fastq.gz | Fastqsanger.gz |
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Project description:Gene expression analysis requires accurate measurements of global RNA degradation rates, earlier problematic with methods disruptive to cell physiology. Recently, metabolic RNA labeling emerged as an efficient and minimally invasive technique applied in mammalian cells. Here, we have adapted SH-Linked Alkylation for the Metabolic Sequencing of RNA (SLAM-Seq) for a global mRNA stability study in yeast using 4-thiouracil pulse-chase labeling. We assign high-confidence half-life estimates for 67.5 % of expressed ORFs, and measure a median half-life of 9.4 min. For mRNAs where half-life estimates exist in the literature, their ranking order was in good agreement with previous data, indicating that SLAM-Seq efficiently classifies stable and unstable transcripts. We then leveraged our yeast protocol to identify targets of the Nonsense-mediated decay (NMD) pathway by measuring the change in RNA half-lives; instead of steady-state RNA level changes. With SLAM-Seq, we assign 580 transcripts as putative NMD targets, based on their measured half-lives in wild-type and upf3Δ mutants. We find 225 novel targets, and observe a strong agreement with previous reports of NMD targets, 61.2 % of our candidates being identified in previous studies.This indicates that SLAM-Seq is a simpler and more economic method for global quantification of mRNA half-lives. Our adaptation for yeast yielded global quantitative measures of the NMD effect on transcript half-lives, high correlation with RNA half-lives measured previously with more technically challenging protocols, and identification of novel NMD regulated transcripts that escaped prior detection.
2022-02-28 | GSE196690 | GEO
Project description:Gene expression analysis requires accurate measurements of global RNA degradation rates, earlier problematic with methods disruptive to cell physiology. Recently, metabolic RNA labeling emerged as an efficient and minimally invasive technique applied in mammalian cells. Here, we have adapted SH-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) for a global mRNA stability study in yeast using 4-thiouracil pulse-chase labeling. We assign high-confidence half-life estimates for 67.5% of expressed ORFs, and measure a median half-life of 9.4 min. For mRNAs where half-life estimates exist in the literature, their ranking order was in good agreement with previous data, indicating that SLAM-seq efficiently classifies stable and unstable transcripts. We then leveraged our yeast protocol to identify targets of the nonsense-mediated decay (NMD) pathway by measuring the change in RNA half-lives, instead of steady-state RNA level changes. With SLAM-seq, we assign 580 transcripts as putative NMD targets, based on their measured half-lives in wild-type and upf3Δ mutants. We find 225 novel targets, and observe a strong agreement with previous reports of NMD targets, 61.2% of our candidates being identified in previous studies. This indicates that SLAM-seq is a simpler and more economic method for global quantification of mRNA half-lives. Our adaptation for yeast yielded global quantitative measures of the NMD effect on transcript half-lives, high correlation with RNA half-lives measured previously with more technically challenging protocols, and identification of novel NMD regulated transcripts that escaped prior detection.
| S-EPMC9074897 | biostudies-literature
Project description:SLAM-seq for K562 endogenous mRNA decay
| PRJNA522262 | ENA
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:Regulation of mRNA stability is a critical step in the control of gene expression. The nonsense mediated mRNA decay (NMD) pathway is one important mRNA stability pathway and has been shown to function in regulating a significant portion of the transcriptome. Perhaps the most significant outstanding question surrounding NMD regulation of endogenous gene expression is the identification of transcripts directly targeted by the NMD pathway versus secondary response genes, whose expression levels are dependent on the activity of direct targets. Here, we present for the first time in an intact animal, the experimental identification of direct targets of NMD. We find that most genes (74%) that are upregulated in NMD mutants appear not to be direct targets of NMD. More surprisingly, we find that the vast majority (97%) of candidate direct targets are not found to be increased in expression in a NMD mutant background. These results imply negative feedback renormalizes the levels of most direct targets of NMD, suggesting they would have been missed in previous analyses. We find that our candidate direct target genes have disproportionately long 3’ UTRs compared to non-targeted control genes. Long 3’ UTRs have been shown to target mRNAs for NMD in experimental systems, and our results imply this is the case in vivo. Our results will allow for the understanding of the function of the NMD pathway in endogenous gene regulation, during normal development, as well as in pathological states. Starting with a Nonsense Mediated Decay(NMD) mutant, in which expression levels of both primary and secondary NMD targets should be increased, we introduce a wild-type copy of the mutated factor and evaluated the transcriptome profile change overtime. Following reintroduction, expression levels of transcripts that are direct targets of NMD should decrease rapidly. Conversely, the levels of indirect targets will remain high until direct targets return to wild-type levels, which will occur later in the timecourse. This allows us to identify direct NMD targets.
2013-07-01 | E-GEOD-47979 | biostudies-arrayexpress
Project description:Transcriptome-wide methodology is developed to capture and sequence decay intermediates deriving from direct NMD targets. Results demonstrate that mammalian-cell NMD generates decay intermediates in a process that involves the covalent addition of nontemplated U, C, G and A nucleotides to 3'-ends. Variations of this methodology demonstrate that these decay intermediates arise co-translationally.
2018-07-05 | GSE111818 | 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:Regulation of mRNA stability is a critical step in the control of gene expression. The nonsense mediated mRNA decay (NMD) pathway is one important mRNA stability pathway and has been shown to function in regulating a significant portion of the transcriptome. Perhaps the most significant outstanding question surrounding NMD regulation of endogenous gene expression is the identification of transcripts directly targeted by the NMD pathway versus secondary response genes, whose expression levels are dependent on the activity of direct targets. Here, we present for the first time in an intact animal, the experimental identification of direct targets of NMD. We find that most genes (74%) that are upregulated in NMD mutants appear not to be direct targets of NMD. More surprisingly, we find that the vast majority (97%) of candidate direct targets are not found to be increased in expression in a NMD mutant background. These results imply negative feedback renormalizes the levels of most direct targets of NMD, suggesting they would have been missed in previous analyses. We find that our candidate direct target genes have disproportionately long 3’ UTRs compared to non-targeted control genes. Long 3’ UTRs have been shown to target mRNAs for NMD in experimental systems, and our results imply this is the case in vivo. Our results will allow for the understanding of the function of the NMD pathway in endogenous gene regulation, during normal development, as well as in pathological states.
2013-07-01 | GSE47979 | GEO
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