HITS-CLIP analysis of FUS in lineage-committed cells.

In the present study, we performed HITS-CLIP analysis for FUS using mouse brain to extensively characterize tits RNA-binding sites and functional roles in RNA metabolisms. We identified preferential binding of FUS to stem-and-loop structures but without any discernible consensus motifs. FUS was preferentially bound to introns and 3' untranslated regions, but the exon/intron boundaries were mostly devoid of FUS-tags. Analysis of position-dependence of FUS-binding sites in regulating inclusion and skipping of exons disclosed that FUS is bound broadly around the alternatively spliced exons. Among them, however, noticeable CLIP-tags were observed in the downstream introns. We also noticed that FUS occasionally binds to the antisense strands in the promoter regions. Global analysis of CLIP-tags and expression profiles revealed that binding of FUS to the promoter antisense regions downgregulates transcription of the sense strand. Overall design: HITS-CLIP (High Throughput Sequencing after Crosslinking and Immunoprecipitation) experiments targeting FUS in mouse cerebrums derived from 12-week-old C57BL/6 mice