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Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3’ UTRs

ABSTRACT: The RNA helicase UPF1 is best known for its key function in mRNA nonsense-mediated mRNA decay (NMD), but has also been implicated in additional mRNA turnover mechanisms, telomere homeostasis, and DNA replication. In NMD, UPF1 recruitment to target mRNAs is thought to occur through interaction with release factors at terminating ribosomes, but evidence for translation-independent interaction of UPF1 with the 3’ untranslated region (UTR) of mRNAs has also been reported. To map UPF1 binding sites transcriptome-wide, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) in human cells, untreated or after inhibiting translation by puromycin. We found a strong association of UPF1 with 3’ UTRs in undisturbed, translationally active cells and a significant increase in UPF1 binding to coding sequence (CDS) after translation inhibition. These results indicate that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. This evidence for translation-independent UPF1-RNA interaction, which is corroborated by RNA immunoprecipitations experiments and by our observation that UPF1 also crosslinks to long non-coding RNAs, suggests that the decision to trigger NMD occurs after association of UPF1 with the mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination.

ORGANISM(S): Homo sapiens

PROVIDER: GSE47976 | GEO | 2013/06/20

SECONDARY ACCESSION(S): PRJNA208516

REPOSITORIES: GEO

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Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3M-bM-^@M-^Y UTRs

Project description:The RNA helicase UPF1 is best known for its key function in mRNA nonsense-mediated mRNA decay (NMD), but has also been implicated in additional mRNA turnover mechanisms, telomere homeostasis, and DNA replication. In NMD, UPF1 recruitment to target mRNAs is thought to occur through interaction with release factors at terminating ribosomes, but evidence for translation-independent interaction of UPF1 with the 3M-bM-^@M-^Y untranslated region (UTR) of mRNAs has also been reported. To map UPF1 binding sites transcriptome-wide, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) in human cells, untreated or after inhibiting translation by puromycin. We found a strong association of UPF1 with 3M-bM-^@M-^Y UTRs in undisturbed, translationally active cells and a significant increase in UPF1 binding to coding sequence (CDS) after translation inhibition. These results indicate that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. This evidence for translation-independent UPF1-RNA interaction, which is corroborated by RNA immunoprecipitations experiments and by our observation that UPF1 also crosslinks to long non-coding RNAs, suggests that the decision to trigger NMD occurs after association of UPF1 with the mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination. Examination of Upf1 binding preferences via iCLIP in untreated HeLa cells and HeLa cells, where translation is blocked by puromycin treatment in vivo crosslinking and immunoprecipitation strategy (iCLIP)

2013-06-20 | E-GEOD-47976 | biostudies-arrayexpress

Quantitative proteomics of affinity purified yeast NMD complexes

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

Ribosome-bound Upf1 forms distinct 80S complexes and conducts mRNA surveillance

Project description:Upf1, Upf2, and Upf3 are the central regulators of nonsense-mediated mRNA decay (NMD), the eukaryotic mRNA quality control pathway generally triggered when a premature termination codon is recognized by the ribosome. The NMD-related functions of the Upf proteins likely commence while these factors are ribosome-associated, but little is known of the timing of their ribosome binding, their specificity for ribosomes translating NMD substrates, or the nature and role of any ribosome:Upf complexes. Here, we have elucidated details of the ribosome-associated steps of NMD. By combining yeast genetics with selective ribosome profiling and co-sedimentation analyses of polysomes with wild-type and mutant Upf proteins, our approaches have identified distinct states of ribosome:Upf association. All three Upf factors manifest progressive polysome association as mRNA translation proceeds, but these events appear to be preceded by formation of a Upf1:80S complex as mRNAs initiate translation. This complex is likely executing an early mRNA surveillance function.

2022-10-21 | PXD029577 | Pride

Ribosome-bound Upf1 forms distinct 80S complexes and conducts mRNA surveillance

2022-10-21 | GSE186795 | GEO

Target discrimination in nonsense-mediated mRNA decay requires Upf1 ATPase activity (CLIP-Seq)

Project description:RNA quality control pathways serve to get rid of faulty RNAs and therefore must be able to discriminate these RNAs from those that are normal. Here we present evidence that the ATPase cycle of SF1 Helicase Upf1 is required for mRNA discrimination during Nonsense-Mediated Decay (NMD). Mutations affecting the Upf1 ATPase cycle disrupt the mRNA selectivity of Upf1, leading to indiscriminate accumulation of NMD complexes on both NMD target and non-target mRNAs. In addition, two modulators of NMD - translation and termination codon-proximal poly(A) binding protein - depend on Upf1 ATPase to limit Upf1-non-target association. Preferential ATPase-dependent dissociation of Upf1 from non-target mRNAs in vitro suggests that selective release of Upf1 contributes to the ATPase-dependence of Upf1 target discrimination. Given the prevalence of helicases in RNA regulation, ATP hydrolysis may be an underappreciated, yet widely employed, activity in target RNA discrimination. CLIP and RIP-seq against Wild Type and Mutant Upf1 in HEK293-T cell lines

2015-07-06 | E-GEOD-69584 | biostudies-arrayexpress

Target discrimination in nonsense-mediated mRNA decay requires Upf1 ATPase activity (RIP-Seq)

2015-07-06 | E-GEOD-69585 | biostudies-arrayexpress

Target discrimination in nonsense-mediated mRNA decay requires Upf1 ATPase activity (RIP-Seq)

2015-07-06 | GSE69585 | GEO

Target discrimination in nonsense-mediated mRNA decay requires Upf1 ATPase activity (CLIP-Seq)

2015-07-06 | GSE69584 | GEO

Nonsense-mediated mRNA decay relies on "two-factor authentication" by SMG5-SMG7

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

UPF1 helicase orchestrates mutually exclusive interactions with the SMG6 endonuclease and UPF2

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

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