C. elegans FBF iCLIP from spermatogenic and oogenic germlines

PUF RNA-binding proteins control stem cells in diverse species, including mammalian, arthropod, and nematode, in addition to other biological functions. The C. elegans PUF protein FBF serves as a paradigm for metazoan PUFs. FBF is essential for the maintenance of germline stem cells but also regulates the hermpahrodite sperm/oocyte cell fate switch and is critical for the process of spermatogenesis. We have attempted to “disentangle” the different roles of FBF by comparing its targets in spermatogenic and oogenic germlines. To this end, we used FBF iCLIP to learn its binding profile in an adult hermaphrodite germline that is sexually transformed and makes only sperm due to a temperature-sensitive sex-determination mutant. As a control, we analyzed FBF iCLIP data from oogenic germlines at the same temperature. Using a modified peak calling algorithm, we identified FBF binding sites in oogenic animals at 20°C, oogenic animals at 25°C, and spermatogenic animals at 25°C. Oogenic FBF targets were similar at 20°C and 25°C. By contrast, FBF mRNA targets in spermatogenetic animals had a distinct profile, revealing sperm-specific targets that are likely critical for the FBF role in spermatogenesis. Most importantly, we found FBF bound to mRNAs regardless of germline gender. In particular, a group of 22 mRNAs clustered as bound with high frequency in a gender- and temperature-independent manner. These 22 mRNAsencode RNA-binding proteins and stem cell regulators and may be crucial for the FBF role in in stem cell maintenance. We generated FBF-1 and FBF-2 iCLIP datasets from animals with somatic tissues of the same gender but germline tissues of opposite gender. For each FBF, we used an N-terminally FLAG-tagged single copy fbf transgene in a strain lacking the endogenous gene (e.g. tagged FLAG::FBF-1 in an fbf-1 null mutant ). Adult hermaphrodites with a sexually transformed germline that are strictly spermatogenic were obtained using a temperature sensitive gain of function mutation in the fem-3 gene (Barton and Kimble 1987). These fem-3 (ts, gf) mutants are spermatogenic at restrictive temperature (25°C). We therefore introduced FLAG-tagged FBF-1 or FBF-2 into the fem-3(ts,gf) mutant, again removing the corresponding endogenous fbf gene in each strain. We then performed FBF iCLIP from adults that were either oogenic or spermatogenic, both raised at 25 °C. For each strain (each FBF, each germline gender), we processed three biological replicates. Three negative control datasets were produced for each germline gender in parallel by omitting the FLAG antibody.