Lynch K W, Maniatis T
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Genes Dev. 1996 Aug 15;10(16):2089-101. doi: 10.1101/gad.10.16.2089.
The Drosophila doublesex female-specific splicing enhancer consists of two classes of regulatory elements, six 13-nucleotide repeat sequences, and a single purine-rich element (PRE). Here, we show that the Drosophila 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. Individual SR proteins were also recruited to the PRE by Tra and Tra2, but in this case they were SF2/ASF and dSRp30 in HeLa and Drosophila cell extracts, respectively. The binding of Tra2, Tra, and the specific SR proteins to the repeats or the PRE was highly cooperative within each complex. Thus, Tra2, which contains a single RNA binding domain, can recognize distinct sequences in the repeats and the PRE in conjunction with specific SR proteins. These observations show that the protein composition of each complex is determined by the RNA recognition sequence and specific interactions between SR proteins and Tra and Tra2.
果蝇双性基因雌性特异性剪接增强子由两类调控元件、六个13核苷酸重复序列和一个单一的富含嘌呤元件（PRE）组成。在此，我们表明果蝇调控蛋白Transformer（Tra）和Transformer 2（Tra2）将剪接因子SR家族的不同成员招募至重复序列和PRE。在HeLa细胞提取物中，在重复序列上形成的复合物由Tra、Tra2和SR蛋白9G8组成。在果蝇Kc细胞提取物中，Tra和Tra2将SR蛋白RBP1招募至重复序列。这些蛋白质在重复序列上按特定顺序排列，SR蛋白位于每个重复序列的5'端，Tra2位于每个3'端。尽管Tra与重复序列的交联不强，但其存在对于Tra2与重复序列3'端的结合至关重要。单个SR蛋白也被Tra和Tra2招募至PRE，但在这种情况下，在HeLa和果蝇细胞提取物中它们分别是SF2/ASF和dSRp30。在每个复合物中，Tra2、Tra和特定SR蛋白与重复序列或PRE的结合具有高度协同性。因此，可以与特定SR蛋白结合识别重复序列和PRE中不同序列的Tra2仅含有一个RNA结合结构域。这些观察结果表明，每个复合物的蛋白质组成由RNA识别序列以及SR蛋白与Tra和Tra2之间的特异性相互作用决定。
