In vivo, SNBs disappeared upon a temperature shift from 37 to 32°C for 24 h and reformed when the temperature was returned to 37°C for 3 h (particle size and count by microscopy on change in temperature). Depletion of Sam68 or HNRNPD by RNAi in HeLa cells in vivo resulted in the disappearance of SNBs as detected by immunofluorescence, whereas depletion of the three other proteins (HNRNPL, DBC1, and ZNF346) hardly affected the integrity of SNBs. The isoforms of HNRNPD containing exon 7 (p42 and p45) localize in SNBs and function in their formation. A coimmunoprecipitation-based detection assay with FLAG-tagged HNRNPD isoforms indicated that p42 and p45 physically interact with Sam68, HNRNPL, and DBC1. Based on Venus-p45 as the wild-type (WT) Venus-HNRNPD construct, two phenylalanine residues essential for RNA binding in each of RRM1 and RRM2 were mutated to aspartic acid to create mutants RRM1-M (F140D/F142D) and RRM2-M (F225D/F227D), which both failed to localize to SNBs, indicating that RNA binding of HNRNPD through the two RRMs simultaneously is essential for its SNB localization. RRM mutants also almost completely lacked the rescue activity for SNB formation. To examine whether the prion-like property of the HNRNPD prion-like domain is required for SNB formation, the tyrosine residues in the PLD of p45 were mutated to serine to create a partial Y-S mutant (nine tyrosine residues were altered) and a full Y-S mutant (all 18 Tyrs mutated). Both PLD Y-S mutants failed to localize to SNBs and showed significantly reduced rescue activity. FLAG-tagged PLD mutants neither interacted with Sam68 nor cotransfected Venus-tagged p45 itself (physical interaction by coimmunoprecipitation), indicating that the HNRNPD PLD is required for the interaction with Sam68 and for the homodimeric interaction of HNRNPD in SNBs. A substantial DBC1 signal was detected when an essential SNB component (protein localization), either Sam68 or HNRNPD, was knocked down. Furthermore, when HNRNPL was knocked down, the DBC1 signal was detected in nuclear foci distinct from those labeled with Sam68 and HNRNPD (protein localization). Both nuclear foci (the Sam68 substructure and the DBC1 substructure) were sensitive to RNase treatment (enzymatic activity assay). No in vitro results available, LLPS, formation of SNBs may require other factors (PMID:27377249). Confocal fluorescence microscopy of HeLa cells overexpressing N-terminally GFP- fused HNRNPD proteins in vivo showed localization HNRNPD into foci (protein localization), and disappearance of foci on the substitution of tyrosine residues to serines (PMID:28709000).