FAX-RIC enables robust RNA interactome profiling in multicellular organisms in vivo
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Mus Musculus (mouse)
TISSUE(S): Liver
SUBMITTER: 
Yongwoo Na
LAB HEAD: Yongwoo Na
PROVIDER: PXD022023 | Pride | 2020-11-25
REPOSITORIES: Pride
Similar Datasets
Project description:Translation of damaged mRNA can lead to ribosome stalling, thereby producing incomplete proteins toxic to the cell. The mechanism of ribosome-associated quality control (RQC) disassembles stalled ribosomes through the actions of the ASC-1 complex (ASCC). Here, we show that some reagents that chemically damage RNA, such as ultraviolet light (UV), cause ribosome stalling, which leads to accumulation of the ASC-1 complex (ASCC) on stalled ribosomes and stable interaction of the ASCC3 helicase with RNA. In contrast, the ASCC was not similarly affected by emetine or anisomycin-induced ribosome stalling. Our work identified two different types of stalled ribosome. Ribosomes arrested by emetine or anisomycin are transient as they are resolved by the ASCC. Whereas the ASCC fails to split some stalled ribosomes, such as those induced by UV, resulting in long-lived stalled ribosome complexes. We show that ribosome stalling activates the G1/S and G2/M cell cycle checkpoints with long-lived stalled ribosomes causing prolonged checkpoint activation. Thus, the cell adjusts this adaptive survival response to match the nature of the stalled ribosome.
2022-02-07 | PXD023976 | Pride
Project description:Translation of damaged mRNA can lead to ribosome stalling, thereby producing incomplete proteins toxic to the cell. The mechanism of ribosome-associated quality control (RQC) disassembles stalled ribosomes through the actions of the ASC-1 complex (ASCC). Here, we show that some reagents that chemically damage RNA, such as ultraviolet light (UV), cause ribosome stalling, which leads to accumulation of the ASC-1 complex (ASCC) on stalled ribosomes and stable interaction of the ASCC3 helicase with RNA. In contrast, the ASCC was not similarly affected by emetine or anisomycin-induced ribosome stalling. Our work identified two different types of stalled ribosome. Ribosomes arrested by emetine or anisomycin are transient as they are resolved by the ASCC. Whereas the ASCC fails to split some stalled ribosomes, such as those induced by UV, resulting in long-lived stalled ribosome complexes. We show that ribosome stalling activates the G1/S and G2/M cell cycle checkpoints with long-lived stalled ribosomes causing prolonged checkpoint activation. Thus, the cell adjusts this adaptive survival response to match the nature of the stalled ribosome.
2022-02-07 | PXD023878 | Pride
Project description:Translation of damaged mRNA can lead to ribosome stalling, thereby producing incomplete proteins toxic to the cell. The mechanism of ribosome-associated quality control (RQC) disassembles stalled ribosomes through the actions of the ASC-1 complex (ASCC). Here, we show that some reagents that chemically damage RNA, such as ultraviolet light (UV), cause ribosome stalling, which leads to accumulation of the ASC-1 complex (ASCC) on stalled ribosomes and stable interaction of the ASCC3 helicase with RNA. In contrast, the ASCC was not similarly affected by emetine or anisomycin-induced ribosome stalling. Our work identified two different types of stalled ribosome. Ribosomes arrested by emetine or anisomycin are transient as they are resolved by the ASCC. Whereas the ASCC fails to split some stalled ribosomes, such as those induced by UV, resulting in long-lived stalled ribosome complexes. We show that ribosome stalling activates the G1/S and G2/M cell cycle checkpoints with long-lived stalled ribosomes causing prolonged checkpoint activation. Thus, the cell adjusts this adaptive survival response to match the nature of the stalled ribosome.
2022-02-07 | PXD023978 | Pride
Project description:Translation of damaged mRNA can lead to ribosome stalling, thereby producing incomplete proteins toxic to the cell. The mechanism of ribosome-associated quality control (RQC) disassembles stalled ribosomes through the actions of the ASC-1 complex (ASCC). Here, we show that some reagents that chemically damage RNA, such as ultraviolet light (UV), cause ribosome stalling, which leads to accumulation of the ASC-1 complex (ASCC) on stalled ribosomes and stable interaction of the ASCC3 helicase with RNA. In contrast, the ASCC was not similarly affected by emetine or anisomycin-induced ribosome stalling. Our work identified two different types of stalled ribosome. Ribosomes arrested by emetine or anisomycin are transient as they are resolved by the ASCC. Whereas the ASCC fails to split some stalled ribosomes, such as those induced by UV, resulting in long-lived stalled ribosome complexes. We show that ribosome stalling activates the G1/S and G2/M cell cycle checkpoints with long-lived stalled ribosomes causing prolonged checkpoint activation. Thus, the cell adjusts this adaptive survival response to match the nature of the stalled ribosome.
2022-02-07 | PXD023861 | Pride
Project description:Utilisation of RNA-binding proteins (RBPs) is an important aspect of post-transcriptional regulation of viral RNA. Viruses such as influenza A viruses (IAV) interact with RBPs to regulate processes including splicing, nuclear export and trafficking, while also encoding RBPs within their genomes, such as NP and NS1. But with almost 1000 RBPs encoded within the human genome it is still unclear what role, if any, many of these proteins play during viral replication. Using the RNA interactome capture (RIC) technique, we isolated RBPs from IAV infected cells to unravel the RBPome of mRNAs from IAV infected human cells. This led to the identification of one particular RBP, MKRN2, that associates with and positively regulates IAV mRNA. Through further validation, we determined that MKRN2 is involved in the nuclear-cytoplasmic trafficking of IAV mRNA likely through an association with the RNA export mediator GLE1. In the absence of MKRN2, IAV mRNAs accumulate in the nucleus of infected cells, which we suspect leads to their degradation by the nuclear RNA exosome complex. MKRN2, therefore, appears to be required for the efficient nuclear export of IAV mRNAs in human cells.
2024-04-16 | PXD048870 | Pride
Project description:1.0 – 1.3 x 10e8 proliferating Jurkat cells at a density of about 1-1.5 x 10e6 cells/mL were employed per sample. Cells were incubated with 0.5mM DMOG (Cayman Chemical Company, 71210) or an equivalent volume of Dimethyl Sulfoxide (DMSO) (vehicle, Merck 1.02950.0500). The DMSO concentration in the medium was 0.023% v/v. Cells were irradiated or not with 254 nm UV light and subjected to eRIC or RIC to determine the RNA-bound proteome. Data correspond to two biologically independent experiments.
2018-09-03 | PXD010942 | Pride
Project description:1.0 – 1.3 x 10e8 proliferating Jurkat cells at a density of about 1-1.5 x 10e6 cells/mL were employed per sample. Cells were irradiated or not with 254 nm UV light and subjected to eRIC or RIC to determine the RNA-bound proteome. Data correspond to two biologically independent experiments.
2018-09-03 | PXD010941 | Pride
Project description:Here, we integrated high-throughput transcriptome and proteome sequencing to construct a comprehensive protein database for the byssus of Chinese green mussel (Perna viridis), aiming at providing novel insights into the molecular mechanisms of byssal binding to heavy metals.
2019-04-30 | PXD009183 | Pride
Project description:Production, function, and turnover of mRNA are orchestrated by multi-subunit machineries that play a central role in gene expression. Within these molecular machines, interactions with the target mRNA are mediated by RNA-binding proteins (RBPs), and the accuracy and dynamics of these RNA-protein interactions are essential for their function. Here, we show that fission yeast whole cell poly(A)+ RNA-protein crosslinking data provides system-wide information on the organisation and function of the RNA-protein complexes. We evaluate relative enrichment of cellular RBPs on poly(A)+ RNA to identify interactors with high RNA9 binding activity and provide key information about the RNA-binding properties of large multi10 protein complexes, such as the mRNA 3’ end processing machinery (cleavage and polyadenylation factor, CPF) and the RNA exosome. We demonstrate that different functional modules within CPF differ in their ability to interact with RNA. Importantly, we reveal that CPF forms additional contacts with RNA via the Fip1 subunit of the polyadenylation module and two subunits of the nuclease module. In addition, our data highlights the central role of the RNA helicase Mtl1 in RNA degradation by the exosome as mutations in Mtl1 lead to 16 disengagement of the exosome from RNA. We examine how routes of substrate access to the complex are affected upon mutation of exosome subunits. Our results provide important insights into how different components of the exosome contribute to engagement of the complex with substrate RNA. Overall, our data uncover how multi-subunit cellular machineries interact with RNA, on a proteome-wide scale.
2021-09-09 | PXD016741 | Pride
Project description:The field of epitranscriptomics is growing in importance, with chemical modification of RNA being associated with a wide variety of biological phenomena. Mass spectrometry (MS) enables the identification of modified RNA residues within their sequence contexts, by using analogous approaches to shotgun proteomics. We have developed a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), as part of the OpenMS software framework. NASE allows the reliable identification of (modified) RNA sequences from LC-MS/MS data in a high-throughput fashion. For this validation dataset, we generated a sample of human total tRNA from a cellular extract - a complex mixture of highly modified RNAs. This sample was RNase-treated prior to nanoflow LC-MS/MS analysis.
2018-12-31 | PXD012097 | Pride