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Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

ABSTRACT: Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

PROVIDER: PRJNA497814 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8083920.fastq.gz	Fastqsanger.gz
	SRR8083930.fastq.gz	Fastqsanger.gz
	SRR8083940.fastq.gz	Fastqsanger.gz
	SRR8083950.fastq.gz	Fastqsanger.gz
	SRR8083921.fastq.gz	Fastqsanger.gz

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Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

Project description:Stress granules are dynamic non-membrane bound organelles made up of untranslating messenger ribonucleoproteins (mRNPs) that form when cells integrate stressful environmental cues resulting in stalled translation initiation complexes. Although stress granules dramatically alter mRNA and protein localization, understanding these complexes has proven to be challenging through conventional imaging, purification, and crosslinking approaches. We therefore developed an RNA proximity labeling technique, APEX-Seq, which uses the ascorbate peroxidase APEX2 to probe the spatial organization of the transcriptome. We show that APEX-Seq can resolve the localization of RNAs within the cell and determine their enrichment or depletion near key RNA-binding proteins. Matching both the spatial transcriptome using APEX-seq, and the spatial proteome using APEX-mass spectrometry (APEX-MS) provide new insights into the organization of translation initiation complexes on active mRNAs, as well as revealing unanticipated complexity in stress granule contents, and provides a powerful approach to explore the spatial environment of macromolecules.

2018-10-26 | GSE121575 | GEO

Off-the-shelf proximity labeling

Project description:Off-the-shelf proximity labeling

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Proximity RNA Labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules.

Project description:Diverse ribonucleoprotein complexes control mRNA processing, translation, and decay. Transcripts in these complexes localize to specific regions of the cell and can condense into non-membrane-bound structures such as stress granules. It has proven challenging to map the RNA composition of these large and dynamic structures, however. We therefore developed an RNA proximity labeling technique, APEX-seq, which uses the ascorbate peroxidase APEX2 to probe the spatial organization of the transcriptome. We show that APEX-seq can resolve the localization of RNAs within the cell and determine their enrichment or depletion near key RNA-binding proteins. Matching the spatial transcriptome, as revealed by APEX-seq, with the spatial proteome determined by APEX-mass spectrometry (APEX-MS), obtained precisely in parallel, provides new insights into the organization of translation initiation complexes on active mRNAs and unanticipated complexity in stress granule composition. Our novel technique allows a powerful and general approach to explore the spatial environment of macromolecules.

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APEX-seq Captures the Protein-RNA Interaction Patterns of DDX3X and DDX3Y

Project description:Although stress granules (SGs) are composed of a stable core structure, they are surrounded by a dynamic shell with assembly, disassembly, and transitions of proteins and RNA between the core and shell. Different SGs have distinct proteins and RNA constituents, which raises the possibility that different SGs might perform different biological functions. The RNA compositions of DDX3X- or DDX3Y-positive SGs are not known, nor is it known what differential effects DDX3X or DDX3Y may exert on these RNA components. To understand the biological function and the compositional differences between DDX3X- or DDX3Y-positive SGs, we first determined the RNA constituents in these SGs using an adapted Ascorbate-peroxidase (APEX)-based proximity labeling method. APEX converts exogenously supplied biotin-phenol (BP) to biotin-phenoxyl radicals, which in turn covalently labels protein and RNA in nanometers. APEX-mediated biotinylation has been widely used in studies of protein-protein interactions and recently in studying protein-RNA interactions. Here, we applied the APEX-mediated biotinylation in DDX3X and DDX3Y SGs to dissect their different RNA composition.

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Profiling stress-triggered RNA condensation with photocatalytic proximity labeling

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In vivo proximity labeling and sequencing (IPL-seq) for SNRPN70 in 293T-Rex

Project description:This proof-of-principle experiment was designed to demonstrate the feasibility of proximity labeling for RNA–protein interactions

2014-10-24 | GSE55370 | GEO

Analysis of subcellular transcriptomes by RNA proximity labeling with Halo-seq

Project description:We have developed Halo-seq, an RNA proximity labeling method that allows the quantification of subcellular transcriptomes. We have demonstrated the efficacy of Halo-seq here by using it to quantify chromatin-proximal, nucleolar, and cytoplasmic transcriptomes. In Halo-seq, RNA molecules in close proximity to a spatially restricted protein are specifically marked and biotinylated, facilitating their separation from bulk cellular RNA and their quantification.

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Project description:To address specificity of ALaP-seq for PMLwt, we performed genome-wide profiling of genomic regions associated with NLS-APEX or PMLca-APEX. Cells were treated with H2O2 to trigger labeling of chromatin with biotin (H2O2+). Experimental negative control for PMLca (H2O2-), where the H2O2 treatment was omitted, was also analyzed.

2020-01-10 | GSE143362 | GEO

Atlas of Subcellular RNA Localization Revealed by APEX-seq

Project description:We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.

2019-06-20 | GSE116008 | GEO

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