ASSEMBLY OF SPECIFIC SR PROTEIN COMPLEXES ON DISTINCT REGULATORY ELEMENTS OF THE DROSOPHILA DOUBLESEX SPLICING ENHANCER

The Drosophila doublesex female-specific splicing enhancer consists of two classes of regulatory elements, six 13-nucleotide repeat sequence s, and a single purine-rich element (PRE). Here, we show that the Dros ophila regulatory proteins Transformer (Tra) and Transformer 2 (Tra2) recruit different members of the SR family of splicing factors to the repeats and the PRE. The complexes formed on the repeats in HeLa cell extract consist of Tra, Tra2, and the SR protein 9G8. In Drosophila Kc cell extract, Tra and Tra2 recruit the SR protein RBP1 to the repeats . These proteins are arranged in a specific order on the repeats, with the SR protein at the 5' end of each repeat, and Tra2 at each 3' end. Although Tra did not cross-link strongly to the repeats, its presence was essential for the binding of Tra2 to the 3' end of the repeat. In dividual SR proteins were also recruited to the PRE by Tra and Tra2, b ut in this case they were SF2/ASF and dSRp30 in HeLa and Drosophila ce ll extracts, respectively. The binding of Tra2, Tra, and the specific SR proteins to the repeats or the PRE was highly cooperative within ea ch complex. Thus, Tra2, which contains a single RNA binding domain, ca n recognize distinct sequences in the repeats and the PRE in conjuncti on with specific SR proteins. These observations show that the protein composition of each complex is determined by the RNA recognition sequ ence and specific interactions between SR proteins and Tra and Tra2.