Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking
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PROVIDER: PRJNA417050 | ENA |
REPOSITORIES: ENA
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Project description:The spatial organization of RNA within cells is a crucial factor influencing a wide range of biological functions throughout all kingdoms of life. However, a general understanding of RNA localization has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we develop such a method, termed APEX-RIP, which combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking. We demonstrate that, using a single protocol, APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, bulk cytosol, and endoplasmic reticulum (ER), with specificity and sensitivity that rival or exceed those of conventional approaches. We further identify candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Since APEX-RIP is simple, versatile, and does not require special instrumentation, we envision its broad application in a variety of biological contexts.
2017-12-14 | GSE106493 | GEO
Project description: Not available
| S-EPMC5730372 | biostudies-literature
Project description:Intricate structure of the interphase chromocenter revealed by proximity based biotinylation
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Project description:We developed a proximity photo-crosslinking method (Spotlight) with a 4-azido-N-ethyl-1,8-naphthalimide (AzNP) moiety that can be converted to reactive aryl nitrene species using ambient blue light-emitting diode light. Using an AzNP-conjugated HaloTag ligand (VL1), blue light-induced photo-crosslinked products of various HaloTag-conjugated proteins of interest were detected in subcellular spaces in live cells. Using Spotlight, we further identified the host interactome of SARS-CoV-2 nucleocapsid (N) protein, which is essential for viral genome assembly. Mass analysis of the VL1-crosslinked product of N-HaloTag in HEK293T cells showed that RNA-binding proteins in stress granules were exclusively enriched in the cross-linked samples. These results tell that our method can reveal the interactome of protein of interest within a short distance in live cells.
2022-04-04 | PXD027149 | Pride
Project description:During interphase centromeres often coalesce into a small number of chromocenters, which can be visualized as distinct, DAPI dense nuclear domains. Intact chromocenters play a major role in maintaining genome stability as they stabilize the transcriptionally silent state of repetitive DNA while ensuring centromere function. Despite its biological importance, relatively little is known about the molecular composition of the chromocenter or the processes that mediate chromocenter formation and maintenance. To shed light on this, we performed confocal and super resolution microscopy and proximity based biotinylation experiments of three proteins associated with the chromocenter in Drosophila: the centromeric H3 variant dCenpA, the heterochromatin protein HP1a and the speciation factor HMR. Our work revealed an intricate internal structure of the chromocenter suggesting a complex multilayered structure of this intranuclear domain.
2020-01-01 | GSE137194 | GEO
Project description:The cytosol-facing organelle outer membrane (OM) is a layer that separates from but also communicates with cellular compartments for the import and exchange of proteins, metabolites and signaling molecules. OM resident proteins are also the targets of cellular surveillance systems by which the cell controls organelle homeostasis for promoting cell survival under stress. However, discovery of OM proteins and their dynamically interacting partners by traditional approaches can be very challenging. In this study, we developed an outer membrane proximity labeling system (OMPL) using biotin ligase mediated proximity biotinylation to map the proximity proteome of the cytosol-facing mitochondria, chloroplast and peroxisome outer membranes in living Arabidopsis cells. The power of this optimized system is highlighted by the discovery of the cytosolic factors and OM receptor candidates that are potentially involved in protein local translation and translocation, membrane contact sites and organelle quality control. Moreover, this OMPL plants generated in this study can be used for rapid and specific isolation of intact mitochondria and peroxisome. Our findings provide ample ground for further investigations on important questions in organelle biology.
2024-01-31 | PXD039752 | JPOST Repository
Project description:Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity biotinylation enzyme to Protein A. Upon target cell permeabilization, the ProtA-Turbo enzyme can be targeted to proteins of interest using an endogenous antibody against the bait protein. Addition of biotin then triggers bait-proximal protein biotinylation. Biotinylated proteins can subsequently be enriched from crude lysates and identified by mass spectrometry. We demonstrate this workflow by targeting Emerin, the histone modification H3K9me3 and the chromatin remodeler BRG1. Amongst the main findings, our experiments revealed Flywch1 as an essential protein that interacts with H3K9me3-marked centromeric heterochromatin. The ProtA-Turbo enzyme represents an ‘off the shelf’ proximity biotinylation enzyme that facilitates proximity biotinylation experiments in primary cells and can be used to understand how proteins cooperate in vivo and how this contributes to cellular homeostasis and disease.
2021-07-12 | PXD025012 | Pride
Project description:Promiscuous labeling enzymes, such as APEX2 or TurboID, are commonly used in in situ biotinylation studies of subcellular proteomes or protein-protein interactions. Although the conventional approach of enriching biotinylated proteins is widely implemented, in-depth identification of specific biotinylation sites remains challenging, and current approaches are technically demanding with low yields. A novel method to systematically identify specific biotinylation sites for LC-MS analysis followed by proximity labeling showed excellent performance compared with that of related approaches in terms of identification depth with high enrichment power. The systematic identification of biotinylation sites enabled a simpler and more efficient experimental design to identify subcellular localized proteins within membranous organelles. Applying this method to the processing body (PB), a non-membranous organelle, successfully allowed unbiased identification of PB core proteins, including novel candidates. We anticipate that our newly developed method will replace the conventional method for identifying biotinylated proteins labeled by promiscuous labeling enzymes.
2024-05-22 | PXD047979 | Pride
Project description:Intracellular proteome aggregation is a ubiquitous disease hallmark with its composition associated with pathogenicity. Herein, we report on a cell-permeable photosensitizer (P8, Rose Bengal derivative) for selective photo-induced proximity labeling, crosslinking and identification of cellular aggregated proteome. Rose Bengal is identified out of common photosensitizer scaffolds for its unique intrinsic binding affinity to various protein aggregates driven by the hydrophobic effect. Further acetylation permeabilizes Rose Bengal for selective imaging, labeling and crosslinking aggregated proteome in live stressed cells. A combination of photo-chemical, tandem mass spectrometry and protein biochemistry characterizations reveal the complexity in photosensitizing pathways (Type I & II), modification sites and labeling mechanisms. The diverse labeling reaction types result in highly effective enrichment and identification of aggregated proteome. Finally, aggregated proteomics and interaction analyses thereby reveal extensive entangling of proteostasis network components mediated by HSP70 chaperone (HSPA1B) and active participation of autophagy pathway in combating proteasome inhibition. Overall, this work exemplifies the first photo-induced proximity labeling and crosslinking method (namely AggID) to profile intracellular aggregated proteome.
2023-08-30 | PXD044948 | iProX
Project description:In this study we developed a new assay to perform proximity biotinylation in cells without the requirement for genetic manipulation or transfection to fuse a biotin ligase to a protein of interest. We show the specificity by targeting H3K9me3 and performing ChIP-seq for the biotin modification. Targeting IgG was used as a control. By doing so, we identified flywch1 as a new protein binding to H3K9me3 regions, which we validated using ChIP-seq (IgG ChIP was used as a control)
2021-07-09 | GSE169317 | GEO