Illumina sequencing of CRAC data obtained with Upf1 in yeast
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1276507 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:The nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic cytoplasmic surveillance pathway that degrades aberrant mRNAs carrying premature termination codons (PTC). In yeast, the long distance between the stop codon and the poly(A) tail dictates mRNA detection by NMD factors Upf1, Upf2 and Upf3, and trigger mRNA decay, independent of deadenylation. While Upf1 binds a wide range of mRNAs beyond PTC-containing substrates, the mechanism of its recruitment on non-PTC-containing mRNAs is unknown. Using Nanopore sequencing of Upf1-bound mRNAs, we discovered that known PTC-containing NMD targets have long poly(A) tails, confirming that Upf1 binding occurs rapidly prior to deadenylation. Strikingly, the largest category specifically bound to Upf1 consisted of mRNAs without PTC harboring short poly(A) tails, corresponding to well-translated and stable transcripts. We showed that the degradation of this subpopulation depends on the three Upf factors, revealing a hitherto unknown function of Upf1 and NMD cofactors in mRNA decay.
2025-08-01 | GSE299782 | GEO
Project description:Illumina sequencing of CRAC data obtained with Ylr419w
| PRJNA1057187 | ENA
Project description:27 years after the yeast genome sequencing, the function of many ORFs remain unknown. Despite the evolutionary distance between human and yeast, homology with the conserved DEAD-box helicase domains allowed us to list DHX29, DHX36 and DHX57 as three putative homologs of the yeast Ylr419w. Functional studies first linked the Ylr419w protein to the translating ribosome, and cross-linking and analysis of cDNA (CRAC) experiments determined the precise region of Ylr419w in contact with the ribosome. It corresponds to the loop of the h16 helix in the 18S rRNA designing the translation initiation factor DHX29, as the functional homolog of Ylr419w.
2024-01-08 | GSE252043 | GEO
Project description:Illumina sequencing of Upf1-TAP immunoprecipitated transcripts in yeast
| PRJNA1191510 | ENA
Project description:Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway known to degrade mRNAs containing premature termination codons (PTCs). A distance long enough between the stop codon and the poly(A)-binding protein (Pab1) is required for mRNA recognition by the NMD factors Upf1, Upf2 and Upf3. Using Nanopore direct RNA sequencing, we show that PTC-containing NMD targets account for only 6% of Upf1-associated RNA and have long poly(A) tails, indicating that Upf1-binding occurs prior to RNA deadenylation. Conversely, most Upf1-associated mRNAs have short poly(A) tails, lack a PTC and correspond to highly expressed genes. A short poly(A) tail is thus an important feature of NMD targets, redefining the scope of this RNA degradation pathway. We propose a model in which loss of Pab1-binding to short poly(A)-tailed mRNAs impairs translation termination and dictates the recruitment of the NMD machinery, uncovering a hitherto unknown role of NMD in the degradation of these transcripts.
2025-06-13 | GSE283053 | GEO
Project description:Upf1 is a master regulator of nonsense-mediated mRNA decay (NMD), an mRNA surveillance and degradation pathway conserved from yeast to human. In S. cerevisiae, Upf1 exists in two distinct complexes with factors that mediate NMD activation or 5'-3' mRNA degradation. We combined endogenous purifications and biochemical reconstitutions of yeast Upf1 complexes with structural analyses and biochemical assays to elucidate the molecular mechanisms driving the mutually exclusive organization of the Upf1-5'-3' and Upf1-2-3 complexes. We show that yeast Upf1 is in a constitutive complex whereby its CH, RecA and C-terminal domains interact with the mRNA decapping factor Dcp2, NMD-associated proteins Nmd4 and Ebs1, and the 5'-3' exoribonuclease Xrn1, respectively. Together, the interacting surfaces and closed conformation of Upf1 in the Upf1-5'-3' complex sterically obstruct the binding of Upf2-3. Our work points to a major restructuring upon recruitment of these factors during NMD and provides insights into evolutionary divergence amongst species.
2025-10-15 | GSE307461 | GEO
Project description:Upf1 is a master regulator of nonsense-mediated mRNA decay (NMD), an mRNA surveillance and degradation pathway conserved from yeast to human. In S. cerevisiae, Upf1 exists in two distinct complexes with factors that mediate NMD activation or 5'-3' mRNA degradation. We combined endogenous purifications and biochemical reconstitutions of yeast Upf1 complexes with structural analyses and biochemical assays to elucidate the molecular mechanisms driving the mutually exclusive organization of the Upf1-5'-3' and Upf1-2-3 complexes. We show that yeast Upf1 is in a constitutive complex whereby its CH, RecA and C-terminal domains interact with the mRNA decapping factor Dcp2, NMD-associated proteins Nmd4 and Ebs1, and the 5' 3' exoribonuclease Xrn1, respectively. Together, the interacting surfaces and closed conformation of Upf1 in the Upf1-5'-3' complex sterically obstruct the binding of Upf2-3. Our work points to a major restructuring upon recruitment of these factors during NMD and provides insights into evolutionary divergence amongst species.
2025-10-14 | PXD068369 | Pride
Project description:Nanopore sequencing of Upf1-TAP immunoprecipitated transcripts in yeast
| PRJNA1199386 | ENA
Project description:We report the application of the Cross-Linking and cDNA (CRAC) technique to identify binding sites of RBP95, ribosome assembly factors, on RNAs in vivo. Once sequenced, Rnas associated have been aligned to the yeast genomes and enrichment of specific RNAs has been studied in more details. Specifically apparition of Mutation/deletion in the reads are of interesdt since they mostly corresponds to prceise site of cross-link between bait protein and target RNA. Here RBP95-HTP is mostly bound to 25S rRNA at one partiuclar site and to several snoRNAs, including SNR37, SNR30, SNR190, SNR86 and SNR128.
2022-08-23 | GSE189589 | GEO
Project description:We report the application of the Cross-Linking and cDNA (CRAC) technique to identify binding sites of DBP6, ribosome assembly factors, on RNAs in vivo. Once sequenced, RNAs associated have been aligned to the yeast genomes and enrichment of specific RNAs has been studied in more details. Specifically apparition of Mutation/deletion in the reads are of interesdt since they mostly corresponds to prceise site of cross-link between bait protein and target RNA. Here DBP6-HTP is mostly bound to snoRNAs (SNR30, SNR10, SNR42, SRN3,SRN190 and SNR13) and 25S rRNA around the same binding sites of the identifed snoRNAs.
2022-12-05 | GSE200692 | GEO