RNAs associated with Upf1, Pab1, Rpl16a, Lsm1 proteins in yeast
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae S288C
PROVIDER: GSE160642 | GEO | 2022/10/01
REPOSITORIES: GEO
Similar Datasets
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:To identify high-confidence NMD targets in mouse N2A neuroblastoma cells, we used our established transcriptome-wide RNA sequencing (RNA-seq) methodologies. Through parallel analyses of RNA-seq upon UPF1-knockdown (KD) and RNA immunoprecipitation (RIP-seq) footprinting of p-UPF1-bound RNAs, we identified 1027 high-confidence neuronal NMD targets.
2021-10-25 | GSE180134 | GEO
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
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-69585 | biostudies-arrayexpress
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.
2015-07-06 | GSE69585 | GEO
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.
2015-07-06 | GSE69584 | 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.
2014-08-15 | GSE60045 | GEO
Project description:To determine the direct binding targets of UPF1, we performed RNA-immunoprecipitation sequencing (RIP-seq), in which mRNAs bound to phosphorylated (NMD-active) UPF1 were immunoprecipitated. And then, we analyzed the pUPF1-bound RNAs by RNA sequencing.
2024-05-30 | GSE234827 | 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
Project description:The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited “closed” state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active “open” state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.
2024-05-29 | GSE246484 | GEO