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Multi-omics analysis using antibody-based in situ biotinylation technique reveals the mechanism of Cajal body formation

ABSTRACT: Multi-omics analysis using antibody-based in situ biotinylation technique reveals the mechanism of Cajal body formation

PROVIDER: PRJNA987706 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR25029463_1.fastq.gz	Fastqsanger.gz
	SRR25029463_2.fastq.gz	Fastqsanger.gz
	SRR25029464_1.fastq.gz	Fastqsanger.gz
	SRR25029464_2.fastq.gz	Fastqsanger.gz
	SRR25029465_1.fastq.gz	Fastqsanger.gz

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Multi-omics analysis using antibody-based in situ biotinylation technique reveals the mechanism of Cajal body formation

Project description:Membrane-less subcellular compartments such as cytoplasmic bodies, nuclear bodies, and paraspeckles play important roles in a variety of cellular functions. Although many of the techniques for identifying the components of cellular bodies were developed to address their function, no technique to comprehensively identify the components of these bodies in both static and dynamic states has been established. Here, we developed an antibody-based in situ biotinylation proximity-labeling technique and succeeded in identifying the components of both static and dynamic nuclear bodies, nucleolus and γH2AX foci, respectively. Using this technique, we comprehensively identified the components of Cajal bodies (CBs), including DNAs, RNAs, and proteins, and clarified their interactome in CBs. Furthermore, we captured the dynamic change of CBs by inhibiting transcription. Our analysis revealed that nascent snRNAs transcribed in CBs play a role in CB nucleation through assembling RNA binding proteins, including FTD-related proteins, RBMs and HNRNPs.

2024-10-01 | GSE235853 | GEO

Multi-omics analysis using antibody-based in situ biotinylation technique reveals the mechanism of Cajal body formation

2024-07-06 | PXD043574 | JPOST Repository

Identification of coilin interactors reveals coordinated control of Cajal body number and substructure

Project description:The cell nucleus contains distinct biomolecular condensates that form at specific genetic loci, organize chromosomes in 3D space, and regulate RNA processing. Among these, Cajal bodies (CBs) require key “scaffolding” proteins for their assembly, which is not fully understood. Here we employ proximity biotinylation, mass spectrometry, and functional screening to comprehensively identify and test functions of CB components. We document 144 protein interactors of coilin, of which 70 were newly detected, and establish 25 players needed for CB assembly and/or maintenance. Surprisingly, depletion of nine coilin interactors – mostly constituents of the 60S ribosome (RPLs) – increased CB number and caused subdomains defined by coilin and the Survival of Motor Neuron protein (SMN) to merge. These phenotypes were traceable to altered nuclear levels of dimethylarginine. Our data implicate RPL24 and other players in the regulation of CBs by modulating post-translational modifications. Moreover, the prevalence of transcription factors among the identified components highlights roles for gene activity in CB assembly and nuclear positioning.

2024-11-10 | PXD042406 | Pride

Identification of the Tau aggresome in progressive supranuclear palsy by in situ biotinylation by antibody recognition

Project description:Here, we used an unbiased proteomic approach, which combines antibody-mediated proximity-detection of co-aggregated proteins together with mass spectrometry. Biotinylation by antibody recognition (BAR) is a recently developed method, by which a primary antibody recognises the target of interest in fixed samples. A secondary antibody conjugated to horseradish peroxidase (HRP) recognises the primary antibody, and with the addition of biotin phenol and hydrogen peroxide, facilitates biotinylation of proteins in close proximity. Biotinylated proteins are then biochemically isolated and identified using mass spectrometry (MS).

2023-02-27 | PXD028237 | Pride

In Situ Proximity Labeling Identifies Lewy Pathology Molecular Interactions in the Human Brain

Project description:The intracellular misfolding and accumulation of alpha-synuclein into structures collectively called LP is a central phenomenon for the pathogenesis of synucleinopathies including Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB). Understanding the molecular architecture of LP is crucial for understanding synucleinopathy disease origins and progression. Here we used a technique called biotinylation by antibody recognition (BAR) to label total (BAR-SYN1) and pathological alpha-synuclein (BAR-PSER129) in situ for subsequent mass spectrometry analysis. This technique has broad potential to help understand the phenomenon of LP in primary human tissue and animal models.

2022-02-22 | PXD028830 | Pride

HVJ-E links Apolipoprotein d to the systemic anti-tumor effects

Project description:Identification of proteins in close proximity to GFP-Apod proteins using in situ biotinylation technique in Tet GFP-Apod B16F10 cells.

2024-06-29 | PXD043386 | JPOST Repository

Cajal bodies are linked to genome conformation

Project description:Cajal bodies are linked to genome conformation

| PRJNA312097 | ENA

Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking

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

Facilitating in situ cross-linking and mass spectrometry by antibody-based protein enrichment

Project description:Cross-linking of living cells followed by mass spectrometry identification of cross-linked peptides (in situ CLMS) is an emerging technology to study protein structures in their native environment. One of the inherent difficulties of this approach is the high complexity of the samples following cell lysis. This difficulty largely limits the identification of cross-links to the more abundant proteins in the cell. Here, we describe a targeted approach in which an antibody pulls a specific protein-of-interest out of the lysate. Mass spectrometry analysis of the protein material that binds to the antibody can then identify considerably more cross-links on the antibody target and its interactors. By using an antibody against the CCT chaperonin, we obtained over two hundred cross-links that provide in situ evidence for the subunit arrangement of CCT and its main interactions with prefoldin. Antibodies against tubulin likewise provided in situ evidence for the structure of the microtubule including the seam. Finally, the approach was also successful in identifying cross-links on a protein expressing at very low amounts (tau in non-neuronal cells). These results demonstrate the general applicability of antibody-based sample simplification for in situ CLMS.

2022-06-09 | PXD025099 | Pride

Phosphorylation of Cajal body protein coilin activated by UHMK1 influences the sensitivity of colon cancer cells to 5-FU

Project description:Drug resistance to 5-fluorouracil (5-FU) and recurrence after chemotherapy in colorectal cancer remain a challenge to be resolved for the improvement of patient outcomes. In the present study, we found that the application of conditioned medium (CM) derived from 5-FU-resistant colon cancer cells HCT-8/FU reduced the chemosensitivity of native HCT-8 to 5-FU, which was accompanied with the significant changes at number counts and morphology of Cajal bodys (CBs), a spherical nuclear body, dynamic exchanging constituent macromolecules (i.e., body-specific proteins and RNAs) with the nucleoplasmic space.It was shown that the disassembly and reassembly of CBs regulated by the phosphorylation of coilin, which was significantly activated by UHMK1. These led to a large number of variations of RNA alternative splicing in cells, which could contribute to the cell survival through remodeling of intracellular phenotype and transmitting preadaptive signal to adjacent cells in tumor microenvironment.

2021-03-15 | GSE168888 | GEO

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