NMD-degradome sequencing reveals ribosome-bound intermediates with 3'-end nontemplated nucleotides
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE111818 | GEO | 2018/07/05
REPOSITORIES: GEO
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Project description:NMD-degradome sequencing reveals ribosome-bound intermediates with 3'-end nontemplated nucleotides
| PRJNA438276 | ENA
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:Nonsense-mediated mRNA decay (NMD) has originally been described as a surveillance mechanism to inhibit the expression of mRNAs with truncated open reading frames (ORFs) and to contribute to the fidelity of gene expression. It is now recognized that NMD also controls the expression of physiological genes with “intact” mRNA. Stress can decrease NMD efficiency and thus increase the mRNA levels of physiological NMD targets. As stress can also inhibit translation, the net outcome for shaping the proteome is difficult to predict. We have thus analyzed de-novo protein synthesis in response to NMD inhibition or the induction of mild endoplasmic reticulum (ER) stress by treatment of cells with the reducing agent dithiotreitol (DTT). We combined pulsed azidohomoalanine (AHA) and stable isotope labeling by amino acids in cell culture (SILAC). Labeled proteins were purified by click chemistry-based covalent coupling to agarose beads, trypsinized, fractionated, and analyzed by LC-MS/MS. We find that mild ER stress upregulates the de-novo synthesis of components of all three branches of the unfolded protein response (PERK, IRE1a and ATF6) without increasing eIF2a phosphorylation or impairing of protein translation. In contrast, inhibition of NMD induces de-novo protein synthesis of downstream targets of the PERK and IRE1a pathways but not the ATF6 pathway. These data thus support a model that implicates a positive feedback loop of ER stress inhibiting NMD efficiency which further promotes the ER stress response in a branch-specific manner.
2016-02-22 | PXD002648 | 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: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 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 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:Nonsense-mediated mRNA decay (NMD) is a conserved RNA degradation pathway that is involved in development, resistance to viral infections and evolution. Upf1, a core NMD factor, is associated with a large variety of cellular RNA in complex ribonucleoprotein particles. We characterized NMD complexes previously by affinity purification of tagged protein followed by mass spectrometry. Here, we extend our observations to proteins that are present in NMD complexes, but for whom the interaction with NMD factors is mediated by RNA. We purified tagged versions of Lsm1, Lsm7, Pat1, Dhh1 and Pab1 and assessed the presence and amount of NMD and RNA-related proteins in each purification. As expected from our previously published results, Upf1 was present in these purifications, suggesting novel hypotheses about the role of Pab1 and the Lsm1-7 complexes in RNA degradation.
2023-08-30 | PXD028008 | Pride
Project description:Nonsense-mediated mRNA decay (NMD) can occur independently of some NMD factors such as UPF3B. We have performed total RNA-Seq in HCT116 cells under NMD factor knockout (with CRISPR-Cas9) and/or knockdown (with siRNA transfection) conditions to identify mRNA substrates regulated by different NMD factors. We have also performed RIPiT-Seq to identify the footprints of three compositionally distinct EJCs that contain the following pairs of proteins: MAGOH-EIF4A3, UPF3B-EIF4A3 or CASC3-EIF4A3 in WT HCT116 cells.
2022-04-23 | GSE179843 | GEO
Project description:Nonsense-mediated mRNA decay (NMD) functions to degrade transcripts bearing premature stop codon (PTC) and is a crucial regulator of gene expression. NMD and the UPF3B gene have been implicated as the cause of various forms of intellectual disability (ID) and other neurological symptoms. Here, we reports three patients with global developmental delay carrying hemizygous deletions of the UPF2 gene, another important member of the NMD pathway and direct interacting partner of UPF3B.
2012-12-31 | GSE35420 | GEO