Defining the RNA Interactome by Total RNA-Associated Protein Purification
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ABSTRACT:
PROVIDER: PRJNA490515 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7822430.fastq.gz | Fastqsanger.gz | |||
| SRR7822431.fastq.gz | Fastqsanger.gz | |||
| SRR7822432.fastq.gz | Fastqsanger.gz | |||
| SRR7822433.fastq.gz | Fastqsanger.gz |
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Project description:UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. We sought to develop a technique to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, total RNA-associated protein purification (TRAPP), relies on SILAC labelling to quantify RNA-associated protein recovery in the presence and absence of irradiation. We utilised TRAPP to study alterations in RNA-protein interactions upon exposure to weak acid stress in yeast. In addition, as UV irradiation induces short distance crosslinks between proteins and nucleic acids, the identity of crosslinked amino acids reveals the exact protein-RNA interacting interface. Precise sites of crosslinking at the level of individual amino acids (iTRAPP) were identified following phospho-peptide enrichment combined with a bioinformatic pipeline (Xi).
2019-03-26 | PXD011071 | Pride
Project description:UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. We sought to develop a technique to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, total RNA-associated protein purification (TRAPP), was applied to yeast (S. cerevisiae ) and bacteria (E. coli). In all analyses, SILAC labeling was used to quantify protein recovery in the presence and absence of irradiation. For S.cerevisiae, we also compared crosslinking using 254nm (UVC) irradiation (TRAPP) with 4-thiouracil (4SU) labeling combined with 350nm (UVA) irradiation (PAR-TRAPP). Recovery of proteins not anticipated to show RNA-binding activity was significantly higher in TRAPP compared to PAR-TRAPP. As an example of preferential TRAPP-crosslinking, we tested enolase (Eno1) and demonstrated its strong, but largely sequence independent, binding to RNA in vivo. We speculate that many protein-RNA interactions have biophysical effects on localization and/or accessibility, by opposing or promoting phase separation. Homologous metabolic enzymes showed RNA crosslinking in S. cerevisiae and E. coli, indicating conservation of this property. TRAPP allows alterations in RNA interactions to be followed and we initially analyzed the effects of weak acid stress. This revealed specific alterations in RNA-protein interactions; for example, during late 60S ribosome subunit maturation. Precise sites of crosslinking at the level of individual amino acids (iTRAPP) were identified following phospho-peptide enrichment combined with a bioinformatic pipeline (Xi). TRAPP is quick, simple and scalable, allowing rapid characterization of the RNA-bound proteome in many systems.
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Project description:The RNA binding proteome (RBPome) was previously investigated using UV crosslinking and purification of poly(A)-associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA-associated protein purification (TRAPP) based on 254 nm UV crosslinking and purification of all RNA-protein complexes using silica beads. In a variant approach (PAR-TRAPP), RNAs were labelled with 4-thiouracil prior to 350 nm crosslinking. PAR-TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBPs. In comparison, TRAPP identified many more proteins not expected to bind RNA, and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNAs. Exploiting PAR-TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA-peptide conjugates, using iTRAPP, providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBPome characterization.
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Project description:Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry (RAP-MS), we identify up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrate the SARS-CoV-2 RNA interactome with proteome abundance changes induced by viral infection and link interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrate by genetic perturbation that CNBP and LARP1, two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduces viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2.
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Project description:Many biological processes are regulated by RNA-RNA interactions 1, nonetheless it remains formidable to analyze the entire RNA interactome. We developed a method, MARIO (MApping Rna-rna Interactions in vivO), to map protein-assisted RNA-RNA interactions in vivo. By circumventing the selection for a specific RNA-binding protein 2-5, our approach vastly expands the identifiable portion of the RNA interactome. Using this technology, we mapped the RNA interactome in mouse embryonic stem cells, which was composed of 46,780 RNA-RNA interactions. The RNA interactome was a scale-free network, with several lincRNAs and mRNAs emerging as hubs. We validated an interaction between two hubs, Malat1 and Slc2a3 using single molecule RNA fluorescence in situ hybridization. Base pairing was observed at the interaction sites of long RNAs, and was particularly strong in transposonRNA-mRNA and lincRNA-mRNA interactions. This reveals a new type of regulatory sequences acting in trans. Consistent with their hypothesized roles, the RNA interaction sites were more evolutionarily conserved than other regions of the transcripts. MARIO also provided new information on RNA structures, by simultaneously revealing the footprint of single stranded regions and the spatially proximal sites of each RNA. The unbiased mapping of the protein-assisted RNA interactome with minimum perturbation of cell physiology will greatly expand our capacity to investigate RNA functions. Three (3) ESC samples were treated with one (1) type of antisense oligonucleotides as is described in Kretz M. et al. (Nature. 2013 Jan 10;493(7431):231-5) to show the RNA interaction among specific RNAs and verify the results from MARIO
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