Fig. 4.

Replicative domains of HMGA1a interact with ORC. a Recombinant STREP-tagged full-length HMGA1a (wild-type), the mutant lacking the acidic C-terminal 15 amino acids (ΔC15) and the R6A/ΔC15 mutant were expressed in E. coli and affinity-purified. The Coomassie gel shows the purified proteins. The schemes of the mutants are depicted on the right. b Pull-down experiments were performed with HeLa nuclear extracts and 7 nmol of each recombinant HMGA1a protein. Co-precipitated ORC-proteins were detected by immunoblot analysis. Orc6 also interacted with the ΔC15 mutant but this interaction was abrogated with the AT-hook mutant R6A/ΔC15 (right). Left lane beads only control. c Affinity-purified HMGA1a and HMGA1aΔC15 incubated with HeLaS3 nuclear extracts and then treated with 75U RNase A and buffer control. Co-precipitated ORC components were monitored by immunoblotting with anti-ORC1, Orc2, Orc4 and Orc6 antibodies. d BiFC analysis of Orc1 or Orc6 with HMGA1a mutant proteins. Orc1 and Orc6 were fused to the HA-tagged N-terminal fragment of YFP. Full-length HMGA1a (top row), HMGA1a containing amino acid (aa) residues 1–92 (ΔC15, middle row), or only the C-terminal acidic domain aa93–107 (C15, lower row) of HMGA1a were linked to the Flag-tagged C-terminal fragment of YFP. Single HMGA1a variants and an ORC BiFC construct were co-transfected into HepG2 cells and fluorescence images were acquired 24 h post-transfection. Expression of ORC-fusion proteins was controlled using monoclonal rat antibodies directed against HA-tag; expression of HMGA1a-fusion proteins was controlled using mouse antibodies directed against the Flag-tag. The frequencies of BIFC signals in cells that co-expressed both YFP-fusion proteins are indicated (%). Scale bars 10 μm. e Model of the HMGA1a interaction. The acidic C-terminal domain of HMGA1a and the AT-hook motifs interact with ORC. The interaction between ORC and the AT-hooks is mediated by RNA (blue line)