Saccharomyces cerevisiae
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA315053 | ENA |
REPOSITORIES: ENA
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Project description:Genetic selection coupled to next-generation sequencing reveals structural requirements for tail-anchor targeting to mitochondria
| PRJEB13011 | ENA
Project description:In animal oocytes and early embryos, mRNA poly(A)-tail length strongly influences translational efficiency (TE), but later in development this coupling between tail length and TE disappears. Here, we elucidate how this coupling is first established and why it disappears. Overexpressing cytoplasmic poly(A)-binding protein (PABPC) in frog oocytes specifically improved translation of short-tailed mRNAs, thereby diminishing coupling between tail length and TE. Thus, coupling requires limiting PABPC, implying that in coupled systems longer-tail mRNAs better compete for limiting PABPC. In addition to expressing excess PABPC, post-embryonic cells had two other properties that prevented strong coupling: terminal-uridylation-dependent destabilization of mRNAs lacking bound PABPC, and a regulatory regime wherein PABPC contributes minimally to TE. Thus, these results revealed three fundamental mechanistic requirements for coupling and defined the context-dependent functions for PABPC, in which this protein promotes TE but not mRNA stability in coupled systems and mRNA stability but not TE in uncoupled systems.
2021-07-07 | GSE166544 | GEO
Project description:The import of nuclear transcribed RNAs into mitochondria is an emerging area that presents tremendous opportunity to develop human metabolic therapeutics. However, our knowledge base is quite limited. Much remains to be discovered regarding specific RNA localization and mechanisms of import. In order to identify novel RNAs imported into mitochondria, all RNAs within the mitochondria were characterized using next generation sequencing technology. Several nuclear transcribed RNAs were found within mitochondrial RNA samples, including nuclear ribosomal RNAs, gamma satellite RNA and VL30 retroelement RNA. The presence of these RNAs within mitochondria coupled with RNA sequencing data (RNAseq) from other laboratories investigating mitochondrial RNA processing, lead us to hypothesize that nuclease treatment of mitoplasts is insufficient for removing contaminating cytoplasmic RNAs. In contrast to traditional methodology, mitochondrial import was evaluated by qRT-PCR after stepwise removal of the outer mitochondrial membrane and subsequent lysis of mitochondria. This allowed identification of RNAs lost from the mitochondria with the same kinetics as mtDNA-transcribed RNAs. This approach provided an improved evaluation of nuclear RNA enrichment within mitochondrial membranes in order to characterize nuclease protection and mitochondrial import and identify false-positive detection errors. qRT-PCR results confirmed the presence of VL30 retroelement RNA within mitochondria and question the hypothesis that the RNA component of RNase P is imported. These results illustrate a reliable approach for evaluating the presence of RNAs within mitochondria and open new avenues of investigation relating to mitochondrial RNA biology and in targeting mitochondrial based therapeutics.
2017-10-08 | GSE62170 | GEO
Project description:In the rapidly advancing field of synthetic biology, there is a critical need for technology to discover targeting moieties for therapeutic biologics. We developed INSPIRE-seq, an approach that utilizes a nanobody library and next-generation sequencing to identify nanobodies selected for complex environments. INSPIRE-seq enables the parallel enrichment of immune cell-binding nanobodies that penetrate the tumor microenvironment. Clone enrichment and specificity varies across immune cell subtypes in the tumor, lymph node, and spleen. INSPIRE-seq identified a dendritic cell binding clone that binds PHB2. Single-cell RNA sequencing revealed a connection with cDC1s, and immunofluorescence confirmed nanobody-PHB2 colocalization along cell membranes. Structural modeling and docking studies assisted binding predictions and will guide nanobody selection. In this work, we demonstrate that INSPIRE-seq offers an unbiased approach to examine complex microenvironments and assist in the development of nanobodies, which could serve as active drugs, modified to become drugs, or used as targeting moieties.
2024-01-26 | PXD046363 | Pride
Project description:Short nascent strands purification coupled to next-generation sequencing allowed us to identify replication origins on human genome in an extensive way, by mapping replication origins in 4 different cell types, IMR-90 fibroblasts, hESC H9 cells, iPSC Th Cl-4 cells and HeLa cells. We demonstrated the existence of a cell type-specific reprogrammable signature of the cell identity revealed by specific efficiencies of conserved origin positions and not by the selection of cell-type specific subsets of origins.
2012-07-01 | GSE37757 | GEO
Project description:Open label Phase II study of FOLFIRI + Panitumumab using ultra-selection technology with next generation high sensitivity genotyping of patients with stage IV colorectal cancer refractory to irinotecan without any mutation on KRAS, PIK3Ca, BRAF and NRAS genes detected with highly sensitive techniques.
| 2133417 | ecrin-mdr-crc
Project description:RNA immunoprecipitation coupled with next generation sequencing (RIP-seq) was used to map the HNRNPL-RNA interactome in LNCaP cells.
2016-12-28 | GSE72841 | GEO
Project description:Dampening functional levels of the mitochondrial deubiquitylating enzyme USP30 has been suggested as an effective therapeutic strategy against neurodegenerative disorders such as Parkinson’s Disease. USP30 inhibition may counteract the deleterious effects of impaired turnover of damaged mitochondria which is inherent to both familial and sporadic forms of the disease. Small-molecule inhibitors targeting USP30 are currently in development, but little is known about their precise nature of binding to the protein. We have integrated biochemical and structural approaches to gain novel mechanistic insights into USP30 inhibition by a small-molecule benzosulfonamide containing compound, USP30inh. Activity-based protein profiling (ABPP) mass spectrometry confirmed target engagement, the high selectivity, and potency of USP30inh for USP30 against 49 other deubiquitylating enzymes in a neuroblastoma cell line. In vitro characterization of USP30inh enzyme kinetics infers slow and tight binding behavior, which is comparable with features of covalent modification of USP30. Finally, we blended hydrogen-deuterium exchange mass spectrometry and computational docking to elucidate the molecular architecture and geometry of USP30 complex formation with USP30inh, identifying structural rearrangements at the cleft of the USP30 thumb and palm subdomains. These studies suggest that USP30inh binds to the thumb-palm cleft that guides the ubiquitin C-terminus into the active site, thereby preventing ubiquitin binding and isopeptide bond cleavage, and confirming its importance in the inhibitory process. Our data will pave the way for the design and development of next-generation inhibitors targeting USP30 and associated deubiquitinylases.
2026-01-12 | PXD042782 | panorama
Project description:We report the MNase-diestion coupled to Next Generation Sequencing of Wild type Drosophila S2 cells or S2 cells over-expressing polycomb protein PH
2022-02-12 | GSE181967 | GEO