Labourier E, Allemand E, Brand S, Fostier M, Tazi J, Bourbon H M
Institut de Génétique Moléculaire, UMR5535 du CNRS, 1919 Route de Mende, F34293 Montpellier Cedex 5, France.
Nucleic Acids Res. 1999 Jun 1;27(11):2377-86. doi: 10.1093/nar/27.11.2377.
The Drosophila repressor splicing factor 1 (RSF1) comprises an N-terminal RNA-binding region and a C-terminal domain rich in glycine, arginine and serine residues, termed the GRS domain. Recently, RSF1 has been shown to antagonize splicing factors of the serine/arginine-rich (SR) family and it is, therefore, expected to play a role in processing of a subset of Drosophila pre-mRNAs through specific interactions with RNA. To investigate the RNA-binding specificity of RSF1, we isolated RSF1-binding RNAs using an in vitro selection approach. We have identified two RNA target motifs recognized by RSF1, designated A (CAACGACGA)- and B (AAACGCGCG)-type sequences. We show here that the A-type cognate sequence behaves as an SR protein-dependent exonic splicing enhancer. Namely, three copies of the A-type ligand bind SR proteins, stimulate the efficiency of splicing of reporter pre-mRNAs several fold and lead to inclusion of a short internal exon both in vitro and in vivo. However, three copies of a B-type ligand were much less active. The finding that RSF1 acts as a potent repressor of pre-mRNA splicing in vitro led us to propose that the equilibrium between a limited number of structurally-related general splicing activators or repressors, competing for common or promiscuous binding sites, may be a major determinant of the underlying mechanisms controlling many alternative pre-mRNA process-ing events.
果蝇阻遏剪接因子1（RSF1）由一个N端RNA结合区域和一个富含甘氨酸、精氨酸和丝氨酸残基的C端结构域组成，该结构域被称为GRS结构域。最近，RSF1已被证明可拮抗富含丝氨酸/精氨酸（SR）家族的剪接因子，因此，预计它通过与RNA的特异性相互作用在果蝇前体mRNA的一个子集的加工过程中发挥作用。为了研究RSF1的RNA结合特异性，我们使用体外筛选方法分离了与RSF1结合的RNA。我们鉴定出了两个被RSF1识别的RNA靶基序，分别命名为A（CAACGACGA）型和B（AAACGCGCG）型序列。我们在此表明，A型同源序列表现为一种依赖SR蛋白的外显子剪接增强子。也就是说，三个拷贝的A型配体结合SR蛋白，将报告前体mRNA的剪接效率提高数倍，并在体外和体内导致一个短的内部外显子的包含。然而，三个拷贝的B型配体活性要低得多。RSF1在体外作为前体mRNA剪接的有效阻遏物这一发现使我们提出，有限数量的结构相关的一般剪接激活剂或阻遏物之间的平衡，竞争共同或混杂的结合位点，可能是控制许多前体mRNA可变加工事件的潜在机制的主要决定因素。
