The CD44 alternative v9 exon contains a splicing enhancer responsive to the SR proteins 9G8, ASF/SF2, and SRp20
The CD44 alternative v9 exon contains a splicing enhancer responsive to the SR proteins 9G8, ASF/SF2, and SRp20
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DOI：
10.1074/jbc.m301090200
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发表时间：
2003-08-29
影响因子：
4.8
通讯作者：
Del Gatto-Konczak, F
中科院分区：
文献类型：
--
作者：
Galiana-Arnoux, D;Lejeune, F;Del Gatto-Konczak, F
CD44 基因替代外显子 v8、v9 和 v10 经常被上皮细胞剪接成块。通过转染仅包含这些替代外显子之一的小基因，我们表明它们中的每一个的剪接都受到细胞类型特异性的控制。通过使用外显子 v8 和 v9 内携带短块突变的小基因，我们在每个外显子中检测到候选外显子剪接增强子。这些候选物在体外激活异源转录物的剪接，因此是真正的外显子剪接增强子。我们进一步分析了覆盖相似30个核苷酸的v9外显子剪接增强子。该增强剂可与 HeLa 核提取物中 35 和 20 kDa 的 SR 蛋白进行 UV 交联。通过使用 S100 提取物中的单个重组 SR 蛋白进行 UV 交联，这些蛋白被鉴定为 9G8、ASF/SF2 和 SRp20。使用重组 9G8、ASF/SF2 和 SRp20 进行的 S100 互补研究表明，所有三种蛋白均可在体外激活含有 v9 增强子的异源外显子的剪接； 9G8 获得了最强的激活。 30 个核苷酸增强子的逐渐截短导致剪接激活逐渐减少。我们提出 9G8、ASF/SF2、SRp20 和可能的其他非 SR 蛋白在体内协同激活 v9 外显子剪接。
The CD44 gene alternative exons v8, v9, and v10 are frequently spliced as a block by epithelial cells. By transfecting minigenes containing only one of these alternative exons, we show that splicing of each of them is under cell type-specific control. By using minigenes carrying short block mutations within exons v8 and v9, we detected a candidate exon splicing enhancer in each of these exons. These candidates activated splicing in vitro of a heterologous transcript and are thus true exon splicing enhancers. We analyzed further a v9 exon splicing enhancer covering similar to30 nucleotides. This enhancer can be UV cross-linked to SR proteins of 35 and 20 kDa in HeLa nuclear extract. By using individual recombinant SR proteins for UV cross-linking in S100 extract, these proteins were identified as 9G8, ASF/SF2, and SRp20. S100 complementation studies using recombinant 9G8, ASF/SF2, and SRp20 showed that all three proteins can activate splicing in vitro of a heterologous exon containing the v9 enhancer; the strongest activation was obtained with 9G8. Progressive truncation of the 30-nucleotide enhancer leads to a progressive decrease in splicing activation. We propose that 9G8, ASF/SF2, SRp20, and possibly other non-SR proteins cooperate in vivo to activate v9 exon splicing.