The genomic landscape of PUF binding in stem cells

We determined the genomic landscape of FBF-1 and FBF-2 binding in germline stem cells using iCLIP, a method that allows identification of protein-RNA interactions at high resolution. We first developed reagents to explore the genomic binding landscapes of full-length FBF-1 and FBF-2 in vivo and then used our iCLIP data to test the precision of several commonly used methods for CLIP peak calling. Based on this iCLIP data, we discovered that FBF-1 and FBF-2 have similar global protein-RNA interaction profiles and that they both target conserved cell cycle regulators and lincRNAs. We found that FBF-1 and FBF-2 regulate their targets through canonical as well as unexpected motif sequences. We elucidated the first in vivo crosslink site analysis for a PUF protein from which we precisely determined FBF-1 and FBF-2 binding sites. Taken together, our data provide an updated model of PUF binding in stem cells. Our study also provides new insight on the control of gene expression in stem cells by RNA binding proteins. Overall design: iCLIP was performed on FLAG-tagged FBF-1 and FBF-2 integrated as a single copy into the C. elegans genome. Wild type worms (N2) went through identical protocols as a negative control.