Figure 4.

SAT III RNA coimmunoprecipitates with CENP-C. Two cell lines were used: GFP–CENP-C transfected S2 cells, or S2 cells carrying GFP-only plasmid. Total cell proteins were isolated and used for purification with GFP-TRAP. After the purification, RNA was isolated and converted to cDNA with random hexamer and oligo (dT) primers. (A) PCR with SAT III–specific primers. SAT III coimmunoprecipitates with CENP-C protein, while it is not present in GFP elution. No RT ctrl, control reaction with no reverse transcription. (B) PCR with GFP-specific primers to test whether RNA binding to CENP-C is nonspecific. GFP RNA was not present in CENP-C elution. No RT ctrl, control reaction with no reverse transcription. (C) WB analysis of GFP–CENP-C and GFP-only purification with GFP-TRAP. (D) CENP-C depletion causes SAT III mislocalization from mitotic chromosomes. Control cells were treated with brown RNAi, and show no reduction in CENP-C levels. SAT III RNA was present on all analyzed mitotic spreads (n = 23). The bottom panels show CENP-C–depleted cells with no detectable SAT III signal (57%, n = 21).