The SR splicing factors ASF/SF2 and SC35 have antagonistic effects on intronic enhancer-dependent splicing of the beta-tropomyosin alternative exon 6A
The SR splicing factors ASF/SF2 and SC35 have antagonistic effects on intronic enhancer-dependent splicing of the beta-tropomyosin alternative exon 6A
复制标题
DOI：
10.1093/emboj/16.7.1772
复制
发表时间：
1997-04-01
期刊：
影响因子：
11.4
通讯作者：
ExpertBezancon, A
中科院分区：
文献类型：
--
作者：
Gallego, ME;Gattoni, R;ExpertBezancon, A
鸡β-原肌球蛋白基因的外显子6A和6 B是互斥的，并且以组织特异性方式选择。外显子6A存在于非肌细胞和平滑肌细胞中，而外显子6 B存在于骨骼肌细胞中。在本研究中，我们研究了外显子GA在非肌细胞中识别的机制。先前的报道已经鉴定了外显子6A下游的富含嘧啶的内含子增强子序列（S4）对于外显子6A的5 '-剪接位点识别是必需的。我们在这里表明，用含有该序列的过量RNA预孵育HeLa细胞提取物特异性地抑制外显子6A通过剪接机制的识别。ASF/SF 2通过与增强子序列的特异性相互作用刺激外显子6A间隔，但不通过SR蛋白SC 35或9 G8，令人惊讶的是：SC 35作为外显子6A剪接的抑制剂，因为向HeLa细胞核提取物中加入增加量的SC 35蛋白完全消除了ASF/SF 2外显子6A剪接的刺激作用。我们的结论是，外显子6A的识别在体外取决于ASF/SF 2到SC 35 SR蛋白的比例，总之，我们的结果表明，这些蛋白质的水平或活性的变化可能有助于组织特异性选择的β-原肌球蛋白外显子6A。在支持这一点，我们表明，从骨骼肌组织中分离的SR蛋白是效率较低的外显子6A的刺激比从HeLa细胞中分离的SR蛋白。
Exons 6A and 6B of the chicken beta-tropomyosin gene are mutually exclusive and selected in a tissue-specific manner. Exon 6A is present in non-muscle and smooth muscle cells, while exon 6B is present in skeletal muscle cells, In this study we have investigated the mechanism underlying exon GA recognition in non-muscle cells. Previous reports have identified a pyrimidine-rich intronic enhancer sequence (S4) downstream of exon 6A as essential for exon 6A 5'-splice: site recognition, We show here that preincubation of HeLa cell extracts with an excess of RNA containing this sequence specifically inhibits exon 6A recognition by the splicing machinery, Splicing inhibition by an excess of this RNA can be rescued by addition; of the SR protein ASF/SF2, but not by the SR proteins SC35 or 9G8, ASF/SF2 stimulates exon 6A spacing through specific interaction with the enhancer sequence, Surprisingly: SC35 behaves as an inhibitor of exon 6A splicing, since addition to HeLa nuclear extracts of increasing amounts of the SC35 protein completely abolish the stimulatory effect of ASF/SF2 an exon 6A splicing. We conclude that exon 6A recognition in vitro depends on the ratio of the ASF/SF2 to SC35 SR proteins, Taken together our results suggest that variations in the level or activity of these proteins could contribute to the tissue-specific choice of beta-tropomyosin exon 6A. In support of this we show that SR proteins isolated from skeletal muscle tissues are less efficient for exon 6A stimulation than SR proteins isolated from HeLa cells.