The splicing factors 9G8 and SRp20 transactivate splicing through different and specific enhancers

The activity of the SR protein family of splicing factors in constitutive o r alternative splicing requires direct interactions with the pre-mRNA subst rate. Thus it is important to define the high affinity targets of the vario us SR species and to evaluate their ability to discriminate between defined RNA targets. We have analyzed the binding specificity of the 30-kDa SR pro tein 9G8, which contains a zinc knuckle in addition to the RNA binding doma in (RBD). Using a SELEX approach, we demonstrate that 9G8 selects RNA seque nces formed by GAC triplets, whereas a mutated zinc knuckle variant selects different RNA sequences, centered around a (A/U)C(A/U)(A/U)C motif, indica ting that the zinc knuckle is involved in the RNA recognition specificity o f 908. In contrast, SC35 selects sequences composed of pyrimidine or purine -rich motifs. Analyses of RNA-protein interactions with purified recombinan t 30-kDa SR proteins or in nuclear extracts, by means of UV crosslinking an d immunoprecipitation, demonstrate that 908, SC35, and ASF/SF2 recognize th eir specific RNA targets with high specificity. Interestingly, the RNA sequ ences selected by the mutated zinc knuckle 908 variant are efficiently reco gnized by SRp20, in agreement with the fact that the RED of 908 and SRp20 a re similar. Finally, we demonstrate the ability of 908 and of its zinc knuc kle variant, or SRp20, to act as efficient splicing transactivators through their specific RNA targets. Our results provide the first evidence for coo peration between an RED and a zinc knuckle in defining the specificity of a n RNA binding domain.