The PUF family of RNA binding proteins has a conserved role in maintaining stem cell self-renewal. FBF is a C. elegans PUF that is required to maintain germline stem cells (GSCs). To understand how FBF controls GSCs, we sought to identify is target mRNAs. Briefly, we immunoprecipitated FBF-mRNA complexes from worm extracts and then used microarrays to identify the FBF-associated mRNAs. To focus on germline targets of FBF, we used a FBF-GFP transgene under the control of a germline promoter and we used an anti-GFP antibody to purify FBF-GFP from worm extracts. In parallel, we also processed a strain expressing TUBULIN-GFP in the germline to control for mRNAs that non-specifically co-purify with GFP. We found that FBF associates with >1,000 unique mRNAs and likely controls a broad network of key cellular and developmental regulators.
Overall design
Worm extracts were prepared from synchronized adult C. elegans (24 h after L4 stage) expressing either a rescuing FBF-1-GFP or TUB-GFP transgene under the control of a germline promoter (pie-1). An immoblized anti-GFP antibody was then used to purify the GFP fusion proteins from extracts. RNA was then extracted from the pellets and analyzed on Affymetrix microarrays. Four biological replicates were performed, each consisting of a FBF-GFP and a TUB-GFP sample processed in parallel.
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