Multi-omics analysis using antibody-based in situ biotinylation technique reveals the mechanism of Cajal body formation
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 ?H2AX 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. Overall design: Experiment 1:  Coilin antibody-based in situ biotinylation DNA-seq with control or Coilin siRNA-transfected HeLa cells. D. melanogastor S2 genomic DNA was used as spike-in control.   Experiment 2: Coilin antibody-based in situ biotinylation RNA-seq in HeLa cells. D. melanogastor S2 RNA was used as spike-in control. Experiment 3: Coilin ChIP-seq in HEK293T cells.