Figure 3.

ORC subunits interact with mRNP complex. (A) The results of immunoprecipitation of ras2, β-tubulin and actin mRNAs from the nuclear fraction of S2 cells, performed with the indicated antibodies (IgG and antibody against TRF2 transcription factor were used as a negative control). The results of RNA immunoprecipitation (RIP) are shown as the percentage of input. (B) The association of Xmas-2 and ORC subunits with ras2 mRNA. Biotin-labeled anti-ras2 or mock (anti-GFP RNA) oligonucleotide bound to streptavidin-agarose beads or the beads alone were incubated with nuclear extract, and associated proteins were identified by western blotting. TRF2 was used as a negative control. (C–E) The effect of the RNAi-mediated knockdown of (C) Xmas-2, (D) Orc5 and (E) Orc3 on the association of ORC subunits with ras2 mRNA assayed by the RIP. RIP was performed with the nuclear fraction of S2 cells treated with dsRNA of GFP (control) or dsRNA of Xmas-2, Orc5 or Orc3. Antibodies used for the immunoprecipitations and the levels of proteins in the extract are shown below the panels. Bars indicate the levels of ras2 mRNA precipitated by antibodies from Xmas-2, Orc5 or Orc3 knockdown extracts (gray bars) normalized relative to its levels precipitated from the GFP knockdown extracts taken as unity (black bars). The horizontal gray band indicates the level of ras2 mRNA co-precipitated with IgG. Error bars show standard deviation (SD) for at least three independent experiments. Asterisks indicate that differences from the control are statistically significant at *P < 0.05 or **P < 0.01 (here, and in Figures 4 and 5).