Figure 6.

Pre-rRNA containing 5.8S rRNA and flanking ITS sequences can be co-immunoprecipitated with RISC proteins. Immunoprecipitation was carried out using antibodies against Drosha, Ago2 or Dicer. Co-selected RNAs were analyzed by RT–PCR. (A) The positions of probe sets in pre-rRNA used for RT–PCR reaction are indicated. The expected sizes of PCR product are given. Co-immunoprecipitated RNAs were subjected to reverse transcription with (+RT) or without (–RT) oligonucleotides complementary to different regions of pre-rRNA or tubulin mRNA. RT reactions were used as templates for PCR amplification. The PCR products were analyzed on 2% agarose gels. Input, RNA prepared from 10% of the material used for immunoprecipitation. –AB, control immunoprecipitation experiment without antibody; M, 1 kb plus DNA ladder. The sizes are shown in base pairs. (B and C) RT–PCR detection for pre-rRNAs containing 5.8S/ITS2 and ITS1/5.8S regions, respectively. (D) RT–PCR for 18S/ITS1 region (upper panel) or 28S/3′ETS region (lower panel). (E) RT–PCR for β-tubulin mRNA. (F) Immunoprecipitation was performed using an antibody against a splicing factor (SF3B3), and the precipitated RNA was subjected to RT–PCR for ITS1/5.8S (3′ITS1) or 5.8S/ITS2 (ITS2) regions.