Identification of an RNA binding specificity for the potential splicing factor TLS

The TLS/FUS gene is involved in a recurrent chromosomal translocation in hu man myxoid liposarcomas. We previously reported that TLS is a potential spl icing regulator able to modulate the 5'-splice site selection in an E1A pre -mRNA. Using an in vitro selection procedure, we investigated whether TLS e xhibits a specificity with regard to RNA recognition. The RNAs selected by TLS share a common GGUG motif. Mutation of a G or U residue within this mot if abolishes the interaction of TLS with the selected RNAs, We showed that TLS can bind GCUG-containing RNAs with a 250 nM affinity. By UV cross-linki ng/competition and immunoprecipitation experiments, we demonstrated that TL S recognizes a GGUG-containing RNA in nuclear extracts. Each one of the RNA binding domains (the three RGG boxes and the RNA recognition motif) contri butes to the specificity of the TLS RNA interaction, whereas only RRM and R GG2-3 participate to the E1A alternative splicing in vivo. The specificity of the TLS RNA interaction was also observed using as natural pre-mRNA, the G-rich IVSB7 intron of the beta -tropomyosin pre-mRNA, Moreover, we determ ined that RNA binding specificities of TLS and high nuclear ribonucleoprote in A1 were different. Hence, our results help define the role of the specif ic interaction of TLS with RNA during the splicing process of a pre-mRNA.