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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE235853 | GEO | 2024/10/01

REPOSITORIES: GEO

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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 frontotemporal dementia-related proteins, RBMs and HNRNPs.

2024-07-06 | PXD043574 | JPOST Repository

TDRD6 instructs correct assembly of mRNPs in the chromatoid body to potentiate UPF1 dependent mRNA decay.

2015-12-08 | GSE63948 | GEO

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

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TDRD6 instructs correct assembly of mRNPs in the chromatoid body to potentiate UPF1 dependent mRNA decay.

Project description:RNA sequencing of heterozygote or Tudor domain contian protein 6 (TDRD6) knockout round spermatid cells. Chromatoid bodies (CBs) are germ cell-specific organelles of largely unknown function. CBs harbor various RNA species, RNA-associated proteins and proteins of the tudor domain family such as TDRD6. Proteome analysis of purified CBs revealed components of the nonsense-mediated mRNA decay machinery such as UPF1. TDRD6 is essential for UPF1 localization to CBs, for UPF1-UPF2 interaction, and for assembly of UPFs and other RNA binding proteins into super-complexes. In absence of TDRD6, the association of some mRNAs with UPF1 is impaired, and the long 3â UTR-stimulated but not the exon junction complex-stimulated pathway of NMD is distorted. Reduced association of mRNAs with UPF1 correlated with increased stability and presence in polysome fractions, i.e. enhanced translational activity. Thus, we define CBs as sites of UPF1-dependent mRNA degradation and provide evidence for the requirement for NMD in spermiogenesis. This function of CBs depends on TDRD6-promoted assembly of mRNA decay enzymes within mRNPs. RNA was extracted from quadruplicate samples and libraries generated for sequencing using the NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs) at the Deep Sequencing Group SFB 655, Biotechnology Center of Technische UniversitÃ¤t Dresden. After enrichment and XP bead (Agencourt AMPure Kit; Beckman Coulter, Inc.) purification, quality control was done using Fragment AnalyzerTM (Advanced Analytical). The bar-coded libraries were equimolarly pooled and subjected to 76 bp single-end sequencing on Illumina HiSeq 2000, resulting in an average of 33 million reads per sample.

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Project description:The multivalent binding of CTCF to a variety of DNA sequences is thought to underlie its ability to mediate a large repertoire of cellular functions. CTCF is a 11 zinc-finger (ZF) protein that is anchored to a majority of its target sites via binding to a 20 base-pair core DNA sequence. Yet the diversity of CTCF binding sites (CBS) has not fully been characterized. Here we assessed CTCF occupancy in cultured mouse cortical neurons as a function of neuronal activity and observed that ~ 22 % of CBS lack the consensus CTCF motif. We report that sequence diversity at most of these atypical CBS is not random but involves degeneracy at specific nucleotide positions within the core CTCF position weight matrix (PWM) that likely affect the binding of ZFs 6 and 7. Surprisingly, degeneracy at the same nucleotides not only define most atypical CBS, but also CBS within most gene promoters, and CBS that are dynamically altered following neuronal stimulation, revealing how atypical CTCF binding could affect gene activity. Dynamic CBS across neural differentiation and neuronal stimulation are found both within and outside loop anchors and TADs, indicating that dynamic CBS could regulate gene activity independently of loop anchoring. Finally, we identified a second mode of atypical CTCF binding that defines most tissue-specific CBS. Unlike other atypical CBS, tissue-specific CBS largely bind sequences unrelated to the core CTCF motif and are enriched within the bodies of tissue-specific genes. Overall, these results indicate how CTCF binding at atypical CBS could allow it to dynamically regulate gene activity patterns during differentiation, development, and in response to environmental cues.

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Atypical modes of CTCF binding facilitate tissue-specific and neuronal activity-dependent gene expression states [SMC1_ChIP-seq]

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Nuclear IQGAP1 promotes gastric cancer cell growth by altering the splicing of a cell-cycle regulon in co-operation with hnRNPM

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The 3' Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies’ association with histone locus bodies

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