Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1054031 | ENA |
REPOSITORIES: ENA
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Project description:Eukaryotic gene expression is constantly regulated and controlled by the translation-coupled nonsense-mediated mRNA decay (NMD) pathway. Aberrant translation termination leads to NMD activation and robust clearance of NMD targets via two seemingly independent and redundant mRNA degradation branches. Here, we uncover that the loss of the first SMG5-SMG7-dependent pathway also inactivates the second SMG6-dependent branch, indicating an unexpected functional hierarchy of the final NMD steps. Transcriptome-wide analyses of SMG5-SMG7-depleted cells confirm complete NMD inhibition resulting in massive transcriptomic alterations. The NMD activity conferred by SMG5-SMG7 is determined to varying degrees by their interaction with the central NMD factor UPF1, heterodimer formation and the initiation of deadenylation. Surprisingly, we find that SMG5 functionally substitutes SMG7 and vice versa. Our data support an improved model for NMD execution that features two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity.
2021-05-08 | PXD024747 | Pride
Project description:Mature mRNAs undergo quality control during translation that may lead to RNA degradation by triggering the nonsense mediated decay (NMD) pathway. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction or an intron in the 3'UTR activates NMD. However, many of the features that lead to the activation of this pathway are unclear. UPF1, an RNA helicase, is the core NMD factor. UPF1 forms a multi-protein complex by recruiting a series of factors and other protein complexes in a process that depends on the UPF1 phosphorylation-dephosphorylation cycle. Among the factors recruited by UPF1, SMG5-SMG7 and SMG6 have critical importance in executing NMD. The SMG5-SMG7 heterodimer induces the exonucleolytic degradation of the mRNA, which depends on the recruitment of deadenylation factors. SMG6 has endonucleolytic activity and cleaves the targeted transcript close to the stop codon. The redundancy between the exonucleolytic and endonucleolytic paths to achieve degradation during NMD has been previously reported in the literature. To investigate the apparent redundancy between SMG5-SMG7 and SMG6 activity and to further understand the features that lead to the activation of NMD, we have generated two clones of SMG7 knockout human cells using CRISPR-Cas9. We generated mRNA-Sequencing data for control and both SMG7 KO clones with additional siRNA-mediated knockdown of Luciferase (Luc) as control, SMG5 or SMG6.
2021-05-07 | E-MTAB-9330 | biostudies-arrayexpress
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.
2014-12-01 | GSE61398 | GEO
Project description:Nonsense-mediated mRNA decay (NMD) is a conserved co-translational mRNA surveillance and turnover pathway across eukaryotes. NMD has a central role in degrading defective mRNAs and also regulates the stability of a significant portion of the transcriptome. The pathway is organized around UPF1, an RNA helicase that can interact with several NMD-specific factors. In human cells, degradation of the targeted mRNAs begins with a cleavage event that requires the recruitment of the SMG6 endonuclease to UPF1. Previous studies have identified functional links between SMG6 and UPF1, but the underlying molecular mechanisms have remained elusive. In this work, we used mass spectrometry, structural biology and biochemical approaches to identify and characterize a conserved short linear motif in SMG6 that interacts with the cysteine/histidine-rich (CH) domain of UPF1. Unexpectedly, we found that the UPF1-SMG6 interaction is precluded when the UPF1 CH domain is engaged with another NMD factor, UPF2. Based on cryo-EM data, we propose that the formation of distinct SMG6-containing and UPF2-containing NMD complexes may be dictated by the RNA-binding status of UPF1. Our findings rationalize a key event in the metazoan NMD quality control pathway and progress our understanding of mechanisms regulating activity and guiding substrate recognition by the SMG6 endonuclease.
2024-05-01 | PXD049474 | Pride
Project description:We aimed to study the cleavage sites of nonsense-mediated mRNA decay (NMD) substrates. Therefore, we depleted the major exoribonuclease XRN1 in human cell culture, which degrades the 3' fragments generated by SMG6-mediated endonucleolytic cleavage. Different reporter mRNAs or endogenous NMD targets were investigated and the 3' fragments were cloned, amplified and subsequently subjected to high throughput sequencing.
2014-11-11 | E-MTAB-2907 | biostudies-arrayexpress
Project description:Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA degradation pathway that targets for degradation faulty mRNAs with premature termination codons as well as many physiological mRNAs encoding full-length proteins. Consequently, NMD functions in both, quality control and post-transcriptional regulation of gene expression, and it has been implicated in the modulation of cancer progression. To investigate the role of NMD in cancer, we knocked out SMG7 in the HT1080 human fibrosarcoma cell line. SMG7 is involved in deadenylation-coupled exonucleolytic mRNA decay, one of the two main degradation pathways in mammalian NMD. Genome-wide proteomic and transcriptomic analyses confirmed that NMD is severely compromised in these SMG7-knockout HT1080 cells. We compared the oncogenic properties between the parental, the SMG7-knockout, and a rescue cell line in which we re-introduced both isoforms of SMG7. In parallel, we tested the effect of a drug inhibiting the NMD factor SMG1 on the HT1080 cells to distinguish NMD-dependent effects from putative NMD-independent functions of SMG7. Using cell-based assays as well as a mouse xenograft tumor model, we show that the oncogenic properties of the parental HT1080 cells areseverely compromised when NMD is inhibited. Molecular pathway analysis revealed a strong reduction of the matrix metalloprotease 9 (MMP9) gene expression in NMD-suppressed cells. Since MMP9 expression promotes cancer cell migration and invasion, metastasis and angiogenesis, its downregulation in NMD-suppressed cells explains, at least partially, their reduced tumorigenicity. Collectively, our findings emphasize the therapeutic potential of NMD inhibition for the treatment ofcertain types of cancer.
2023-09-19 | PXD041853 | Pride
Project description:Nonsense-mediated mRNA decay (NMD) controls the quality of eukaryotic gene expression and also degrades physiologic mRNAs. How NMD targets are identified is incompletely understood. A central NMD factor is the ATP-dependent RNA helicase UPF1. Neither the distance in space between the termination codon and the poly(A) tail nor the binding of steady-state, largely hypophosphorylated UPF1 is a discriminating marker of cellular NMD targets, unlike for PTC-containing reporter mRNAs when compared to their PTC-free counterparts. Here, we map phosphorylated UPF1 (p-UPF1) binding sites using transcriptome-wide footprinting or DNA oligonucleotide-directed mRNA cleavage to report that p-UPF1 provides the first reliable cellular NMD-target marker. p-UPF1 is enriched on NMD target 3'UTRs along with SMG5 and SMG7 but not SMG1 or SMG6. Immunoprecipitations of UPF1 variants deficient in various aspects of the NMD process in parallel with FRET experiments reveal that ATPase/helicase-deficient UPF1 manifests high levels of RNA binding and disregulated hyperphosphorylation, whereas wild-type UPF1 releases from nonspecific RNA interactions in an ATP hydrolysis-dependent mechanism until an NMD target is identified. 3'UTR-associated UPF1 undergoes regulated phosphorylation on NMD targets, providing a binding platform for mRNA degradative activities. p-UPF1 binding to NMD target 3'UTRs is stabilized by SMG5 and SMG7. Our results help to explain why steady-state UPF1 binding is not a marker for cellular NMD substrates and how this binding is transformed to induce mRNA decay.
2014-08-15 | GSE60045 | GEO
Project description:Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA degradation pathway that targets for degradation faulty mRNAs with premature termination codons as well as many physiological mRNAs encoding full-length proteins. Consequently, NMD functions in both, quality control and post-transcriptional regulation of gene expression, and it has been implicated in the modulation of cancer progression. To investigate the role of NMD in cancer, we knocked out SMG7 in the HT1080 human fibrosarcoma cell line. SMG7 is involved in deadenylation-coupled exonucleolytic mRNA decay, one of the two main degradation pathways in mammalian NMD. Genome-wide proteomic and transcriptomic analyses confirmed that NMD is severely compromised in these SMG7-knockout HT1080 cells. We compared the oncogenic properties between the parental, the SMG7-knockout, and a rescue cell line in which we re-introduced both isoforms of SMG7. In parallel, we tested the effect of a drug inhibiting the NMD factor SMG1 on the HT1080 cells to distinguish NMD-dependent effects from putative NMD-independent functions of SMG7. Using cell-based assays as well as a mouse xenograft tumor model, we show that the oncogenic properties of the parental HT1080 cells are severely compromised when NMD is inhibited. Molecular pathway analysis revealed a strong reduction of the matrix metalloprotease 9 (MMP9) gene expression in NMD-suppressed cells. Since MMP9 expression promotes cancer cell migration and invasion, metastasis and angiogenesis, its downregulation in NMD-suppressed cells explains, at least partially, their reduced tumorigenicity. Collectively, our findings emphasize the therapeutic potential of NMD inhibition for the treatment of certain types of cancer.
2023-09-06 | GSE232333 | GEO
Project description:Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA degradation pathway that targets for degradation faulty mRNAs with premature termination codons as well as many physiological mRNAs encoding full-length proteins. Consequently, NMD functions in both, quality control and post-transcriptional regulation of gene expression, and it has been implicated in the modulation of cancer progression. To investigate the role of NMD in cancer, we knocked out SMG7 in the HT1080 human fibrosarcoma cell line. SMG7 is involved in deadenylation-coupled exonucleolytic mRNA decay, one of the two main degradation pathways in mammalian NMD. Genome-wide proteomic and transcriptomic analyses confirmed that NMD is severely compromised in these SMG7-knockout HT1080 cells. We compared the oncogenic properties between the parental, the SMG7-knockout, and a rescue cell line in which we re-introduced both isoforms of SMG7. In parallel, we tested the effect of a drug inhibiting the NMD factor SMG1 on the HT1080 cells to distinguish NMD-dependent effects from putative NMD-independent functions of SMG7. Using cell-based assays as well as a mouse xenograft tumor model, we show that the oncogenic properties of the parental HT1080 cells are severely compromised when NMD is inhibited. Molecular pathway analysis revealed a strong reduction of the matrix metalloprotease 9 (MMP9) gene expression in NMD-suppressed cells. Since MMP9 expression promotes cancer cell migration and invasion, metastasis and angiogenesis, its downregulation in NMD-suppressed cells explains, at least partially, their reduced tumorigenicity. Collectively, our findings emphasize the therapeutic potential of NMD inhibition for the treatment of certain types of cancer.
2023-09-04 | GSE232185 | GEO
Project description:Nonsense-mediated mRNA decay (NMD) controls the quality of eukaryotic gene expression and also degrades physiologic mRNAs. How NMD targets are identified is incompletely understood. A central NMD factor is the ATP-dependent RNA helicase UPF1. Neither the distance in space between the termination codon and the poly(A) tail nor the binding of steady-state, largely hypophosphorylated UPF1 is a discriminating marker of cellular NMD targets, unlike for PTC-containing reporter mRNAs when compared to their PTC-free counterparts. Here, we map phosphorylated UPF1 (p-UPF1) binding sites using transcriptome-wide footprinting or DNA oligonucleotide-directed mRNA cleavage to report that p-UPF1 provides the first reliable cellular NMD-target marker. p-UPF1 is enriched on NMD target 3'UTRs along with SMG5 and SMG7 but not SMG1 or SMG6. Immunoprecipitations of UPF1 variants deficient in various aspects of the NMD process in parallel with FRET experiments reveal that ATPase/helicase-deficient UPF1 manifests high levels of RNA binding and disregulated hyperphosphorylation, whereas wild-type UPF1 releases from nonspecific RNA interactions in an ATP hydrolysis-dependent mechanism until an NMD target is identified. 3'UTR-associated UPF1 undergoes regulated phosphorylation on NMD targets, providing a binding platform for mRNA degradative activities. p-UPF1 binding to NMD target 3'UTRs is stabilized by SMG5 and SMG7. Our results help to explain why steady-state UPF1 binding is not a marker for cellular NMD substrates and how this binding is transformed to induce mRNA decay. RIP-seq experiments for p-UPF1, control IPs using rabbit IgG and additional control sample without IP were performed in duplicates
2014-08-15 | E-GEOD-60045 | biostudies-arrayexpress