RIP-Chip analysis of the C. elegans FOG-1 and FOG-3 proteins

FOG-1/CPEB and FOG-3/Tob are the terminal regulators of the sex determination in C. elegans germ cells. CPEB and Tob proteins are both translational regulators. To investigate how FOG-1 and FOG-3 regulate germ cell sex determination we sought to identify the target mRNAs. We used transgenic epitope tagged animals (3xMyc::FOG-1 and FOG-3::3xFLAG). To identify the mRNA targets of FOG-1/CPEB and FOG-3/Tob on a genome wide scale we used RNA immunoprecipitation followed by microarray analysis. We found 81 putative mRNA targets of FOG-1 and 722 putative targets of FOG-3. 76 target mRNAs were common to both FOG-1 and FOG-3. FOG-1 RNA-coimmunoprecipitation (RIP) samples were prepared as follows. Parallel RIPs were performed with wild type animals as the control IP. Worm extracts were made from wildtype and 3xMyc::FOG-1 late L3/early L4 animals. Myc affinity gel was used to immunoprecipitate 3xMyc::FOG-1 from the extracts. RNA was extracted from the pellets and analyzed on Affymetrix microarrays. 7 biological replicates of both 3xMyc::FOG-1 and wildtype were used. FOG-3 RNA-coimmunoprecipitation (RIP) samples were prepared as follows. Parallel RIPs were performed with wild type animals as the control IP. Worm extracts were made from wildtype and FOG-3::3xFLAG late L3/early L4 animals. Myc affinity gel was used to immunoprecipitate FOG-3::3xFLAG from the extracts. RNA was extracted from the pellets and analyzed on Affymetrix microarrays. 7 biological replicates of both FOG-3::3xFLAG and wildtype were used.