NMD-degradome sequencing reveals ribosome-bound intermediates with 3'-end nontemplated nucleotides
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ABSTRACT:
PROVIDER: PRJNA438276 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6833430_1.fastq.gz | Fastqsanger.gz | |||
| SRR6833430_2.fastq.gz | Fastqsanger.gz | |||
| SRR6833431_1.fastq.gz | Fastqsanger.gz | |||
| SRR6833431_2.fastq.gz | Fastqsanger.gz | |||
| SRR6833432_1.fastq.gz | Fastqsanger.gz |
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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:Nonsense-mediated messenger RNA decay (NMD) controls mRNA quality and degrades physiologic mRNAs to fine-tune gene expression in changing developmental or environmental milieus. NMD requires that its targets are removed from the translating pool of mRNAs. Since the decay steps of mammalian NMD remain unknown, we developed assays to isolate and sequence direct NMD decay intermediates transcriptome-wide based on their co-immunoprecipitation with phosphorylated UPF1, which is the active form of this essential NMD factor. We show that, unlike steady-state UPF1, phosphorylated UPF1 binds predominantly deadenylated mRNA decay intermediates and activates NMD cooperatively from 5'- and 3'-ends. We leverage method modifications to characterize the 3'-ends of NMD decay intermediates, show that they are ribosome-bound, and reveal that some are subject to the addition of non-templated nucleotide. Uridines are added by TUT4 and TUT7 terminal uridylyl transferases and removed by the Perlman syndrome-associated exonuclease DIS3L2. The addition of other non-templated nucleotides appears to inhibit decay.
| S-EPMC8262411 | biostudies-literature
Project description:Exon junction complexes (EJCs) are deposited to mRNAs during splicing and displaced from mRNAs by ribosomes in the pioneer round of translation. The understanding of EJC-bound mRNA degradation before steady-state translation has been limited to nonsense-mediated mRNA decay (NMD) due to a lack of suitable methodologies. Here, we show that RNA degradome data of Arabidopsis, rice, worm and human cells all exhibit a predominant accumulation of 5′ monophosphate (5′P) ends in the canonical EJC region. Inhibition of 5′-3′ exoribonuclease activity and overexpression of an EJC disassembly factor in Arabidopsis reduced the 5′P ends accumulating in the EJC region, supporting the notion that these 5′P ends are in vivo EJC footprints. Hundreds of Arabidopsis NMD targets possess evident EJC footprints, validating their degradation during the pioneer round of translation. In addition to premature termination codons, plant microRNAs can also direct the degradation of EJC-bound mRNAs. However, the production of EJC footprints from NMD but not microRNA targets is dependent on an NMD factor, SMG7. Together, our finding demonstrating in vivo EJC footprinting unravels the composition of the RNA degradome, and provides a new avenue for studying NMD and other mechanisms, such as microRNAs, targeting EJC-bound mRNAs for degradation before steady-state translation.
2020-02-02 | GSE118215 | GEO
Project description:In metazoans, the endoribonuclease SMG6 is thought to cleave many endogenous mRNAs targeted for nonsense-mediated mRNA decay (NMD). However, most evidence as to the identity of endogenous SMG6 substrates is indirect, and little is known about their cleavage sites. Here, we report the efficacy of an RNA degradome approach called parallel analysis of RNA ends (PARE) for identifying NMD intermediates in human cells. By specifically sequencing the 5’ ends of intermediates dependent on SMG6 and the critical NMD factor UPF1, hundreds of endogenous transcripts that are direct targets of SMG6 have been revealed. A preferred sequence motif spanning most SMG6 cleavage sites has been identified and validated by mutational analysis. For many SMG6 substrates, depletion of SMG6 leads to decapping of the RNAs. These findings provide key insights into NMD and targeting by SMG6 while also demonstrating the potential of PARE for analyzing other ribonucleases with diverse endogenous substrates.
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Project description:To identity the targets of miRNAs, we bundled 12 samples from different developing satages into four mixture samples. These samples were used to cosntruct degradome libraries and preform degradome sequencing on Illumina Hi-seq 2000 analyzer. More than 44.98 millions clean reads were obtained and 33.52 million reads were mapped to the soybean cDNA. The mapped reads were used to identity miRNA targets by CleaveLand4 pipeline. 4 degradome mixed samples, no replicates, but every degradome data consists of two parts data. Please note that every degradome sample has two processed and two raw data files. To have enough data, additional sequencing was performed from each sample library. And each sample raw data was processed separately (tissue_name*degradome.txt) and also combined (all_degradome*.txt).
2017-05-01 | E-GEOD-72903 | biostudies-arrayexpress
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.
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