FIGURE 4.

RIP-Chip identifies endogenous STAU-1-associated mRNAs. A, scheme for identification of STAU-1-associated mRNAs. α-STAU-1 antibody was used to immunoprecipitate endogenous STAU-1 from wild-type young adult lysate (WT IP). Two negative controls included immunoprecipitation using preimmune sera with wild-type young adult lysate (Preimmune IP) and immunoprecipitation using α-STAU-1 antibody with stau-1(ΔdsRBD4) young adult lysate (Mutant IP). RNA was isolated from the immunoprecipitations, linearly amplified, and hybridized to Affymetrix C. elegans genome arrays. The mRNAs associated with the WT IP were compared with those associated with the mutant IP (Comparison 1) and the Preimmune IP (Comparison 2) to identify STAU-1-associated mRNAs. B, Western analysis shows the α-STAU-1 antibody specifically immunoprecipitates STAU-1 protein from wild-type lysate (lane 2, top) but does not immunoprecipitate mutant protein (STAU-1(ΔdsRBD4)) in which the epitope is deleted (lane 5, top). STAU-1 protein is also not immunoprecipitated with preimmune serum (lane 3, top). α-STAU-1 antibody detects STAU-1 protein in the wild-type input (lane 1, top) but does not detect the mutant protein expressed in the stau-1(ΔdsRBD4) input (lane 4, top). α-Actin served as a loading control for the inputs (lanes 1 and 4, bottom). C, the log mean intensity for each microarray probe (x axis) was determined for preimmune IP (blue line), mutant IP (green line), and WT IP (red line) and plotted against the proportion of probes (y axis) with a given mean intensity. Overall, the WT IP has enriched signal intensity compared with the control IPs (blue and green lines). D, we compared WT IP with preimmune IP and compared WT IP with mutant IP and determined which transcripts had the highest SAM rank and were at least 4-fold enriched in the WT IP. Transcripts meeting these criteria from each comparison overlapped, resulting in 418 common STAU-1-associated mRNAs.