Toward Identifying Subnetworks from FBF Binding Landscapes in Caenorhabditis Spermatogenic or Oogenic Germlines

Metazoan PUF (Pumilio and FBF) RNA-binding proteins regulate various biological processes, but a common theme across phylogeny is stem cell regulation. In Caenorhabditis elegans, FBF (fem-3 Binding Factor) maintains germline stem cells regardless of which gamete is made, but FBF also functions in the process of spermatogenesis. We have begun to disentangle these biological roles by asking which FBF targets are gamete-independent, as expected for stem cells, and which are gamete-specific. Specifically, we compared FBF iCLIP binding profiles in adults making sperm to those making oocytes. Normally, XX adults make oocytes. To generate XX adults making sperm, we used a fem-3(gf) mutant requiring growth at 25 degrees; for comparison, wild-type oogenic hermaphrodites were also raised at 25 degrees. Our FBF iCLIP data revealed FBF binding sites in 1522 RNAs from oogenic adults and 1704 RNAs from spermatogenic adults. More than half of these FBF targets were independent of germline gender. We next clustered RNAs by FBF-RNA complex frequencies and found four distinct blocks. Block I RNAs were enriched in spermatogenic germlines, and included validated target fog-3, while Block II and III RNAs were common to both genders, and Block IV RNAs were enriched in oogenic germlines. Block II (510 RNAs) included almost all validated FBF targets and was enriched for cell cycle regulators. Block III (21 RNAs) was enriched for RNA-binding proteins, including previously validated FBF targets gld-1 and htp-1. We suggest that Block I RNAs belong to the FBF network for spermatogenesis, and that Blocks II and III are associated with stem cell functions.