RIP-chip analysis of the C. elegans PUF protein FBF

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.PUF家族RNA结合蛋白（RNA binding proteins）在维持干细胞自我更新过程中具有保守的生物学功能。FBF是秀丽隐杆线虫（C. elegans）的PUF家族蛋白，是维持生殖系干细胞（germline stem cells, GSCs）所必需的因子。为解析FBF调控生殖系干细胞的分子机制，我们着手鉴定其靶信使RNA（messenger RNA, mRNA）。简言之，我们从线虫提取物中免疫共沉淀FBF-mRNA复合物，随后通过微阵列芯片（microarrays）技术鉴定与FBF结合的mRNA。为聚焦FBF的生殖系靶标，我们使用了受生殖系启动子（germline promoter）调控的FBF-绿色荧光蛋白（green fluorescent protein, GFP）转基因（transgene）品系，并采用抗GFP抗体从线虫提取物中纯化FBF-GFP复合物。与此同时，我们还处理了一株在生殖系中表达微管蛋白-GFP（TUBULIN-GFP）的菌株，作为阴性对照以甄别与GFP非特异性共纯化的mRNA。我们发现，FBF可与超过1000种独特的mRNA结合，并大概率调控一个涵盖关键细胞与发育调控因子的广泛调控网络。