Figure 6. DBC1 is a component of a SNB substructure and a novel RNase-sensitive NB. (A) Structure-based sequence alignment of the S1 domain. The mutated residues are indicated by red arrowheads above the alignment. HsDBC1, Homo sapiens DBC1; BsCSPB, B. subtilis cold shock protein (CSP) B; TtCDP, Thermus thermophilus CSP; NmCPS, Neisseria meningitidis CSP; Ec CSP, Escherichia coli CSP; HsCRHSP-24, H. sapiens CRHSP-24; HsUNR, H. sapiens UNR; DmUNR, D. melanogaster UNR. (B) Structure of the BsCSP-RNA complex (PDB ID: 3pf5) is shown on the left, and the BsCPSB-based homology model of the S1L domain of HsDBC1 is shown on the right. Positions of mutated Phe 69 and Phe 77 are shown. (C) Schematics of WT protein and mutants with the S1L domain deletion (ΔS1L) and the S1L domain point mutations (S1L-M). Amino acid numbers are shown above. (D) The S1L of DBC1 is required for localization to the SNB in HeLa cells. Venus-tagged WT, ΔS1L, and S1L-M were transfected into HeLa cells. Endogenous Sam68 is a marker of the SNB in HeLa cells. The line scan data are shown on the right. (E) The mutation in the S1L domain does not affect expression. Expression of the three DBC1 proteins shown in C was measured by Western blotting. The molecular mass marker (kD) is shown on the left. α-Tubulin is a control. (F) The S1L domain is required for RNA binding of DBC1. UV cross-linking and streptavidin pull-down of FLAG-SBP-tagged WT, ΔS1L, and S1L-M was performed. After RNase digestion, the cross-linked RNA fragments were 32P-labeled with polynucleotide kinase. The radiolabeled DBC1-RNA complexes were separated by SDS-PAGE. IP was confirmed by Western blotting (WB) with an anti-FLAG antibody. The molecular mass marker (kD) is shown on the left. (G) The S1L domain of DBC1 is required for localization to the DBC1 body in HCT116 cells. Venus-tagged WT and ΔS1L were transfected into HCT116 cells. Endogenous DBC1 is a marker of the DBC1 body. The cell populations (%) in which DBC1-Venus and the endogenous DBC1 signals overlap are shown in the merge panels (>100 cells, n = 3). Bars, 10 µm.
Figure 6.

DBC1 is a component of a SNB substructure and a novel RNase-sensitive NB. (A) Structure-based sequence alignment of the S1 domain. The mutated residues are indicated by red arrowheads above the alignment. HsDBC1, Homo sapiens DBC1; BsCSPB, B. subtilis cold shock protein (CSP) B; TtCDP, Thermus thermophilus CSP; NmCPS, Neisseria meningitidis CSP; Ec CSP, Escherichia coli CSP; HsCRHSP-24, H. sapiens CRHSP-24; HsUNR, H. sapiens UNR; DmUNR, D. melanogaster UNR. (B) Structure of the BsCSP-RNA complex (PDB ID: 3pf5) is shown on the left, and the BsCPSB-based homology model of the S1L domain of HsDBC1 is shown on the right. Positions of mutated Phe 69 and Phe 77 are shown. (C) Schematics of WT protein and mutants with the S1L domain deletion (ΔS1L) and the S1L domain point mutations (S1L-M). Amino acid numbers are shown above. (D) The S1L of DBC1 is required for localization to the SNB in HeLa cells. Venus-tagged WT, ΔS1L, and S1L-M were transfected into HeLa cells. Endogenous Sam68 is a marker of the SNB in HeLa cells. The line scan data are shown on the right. (E) The mutation in the S1L domain does not affect expression. Expression of the three DBC1 proteins shown in C was measured by Western blotting. The molecular mass marker (kD) is shown on the left. α-Tubulin is a control. (F) The S1L domain is required for RNA binding of DBC1. UV cross-linking and streptavidin pull-down of FLAG-SBP-tagged WT, ΔS1L, and S1L-M was performed. After RNase digestion, the cross-linked RNA fragments were 32P-labeled with polynucleotide kinase. The radiolabeled DBC1-RNA complexes were separated by SDS-PAGE. IP was confirmed by Western blotting (WB) with an anti-FLAG antibody. The molecular mass marker (kD) is shown on the left. (G) The S1L domain of DBC1 is required for localization to the DBC1 body in HCT116 cells. Venus-tagged WT and ΔS1L were transfected into HCT116 cells. Endogenous DBC1 is a marker of the DBC1 body. The cell populations (%) in which DBC1-Venus and the endogenous DBC1 signals overlap are shown in the merge panels (>100 cells, n = 3). Bars, 10 µm.

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