A 5′ splice site-proximal enhancer binds SF1 and activates exon bridging of a microexon
A 5′ splice site-proximal enhancer binds SF1 and activates exon bridging of a microexon
复制标题
DOI：
10.1128/mcb.20.11.3988-3995.2000
复制
发表时间：
2000-06-01
影响因子：
5.3
通讯作者：
Berget, SM
中科院分区：
文献类型：
--
作者：
Carlo, T;Sierra, R;Berget, SM
在脊椎动物中，内部外显子大小在狭窄的大小范围内发生。实验上，短于50个核苷酸的外显子很少包括在mRNA中，除非伴随有增强的剪接位点或作为剪接增强子的辅助序列，这表明snRNP和同时结合到微外显子的3'和5'剪接位点的其他剪接因子之间的空间干扰。尽管存在这些问题，但存在非常小的天然存在的外显子。在这里，我们研究了识别心肌肌钙蛋白T基因组成性包含的六核苷酸外显子的因素和机制。该外显子的包含依赖于位于5'剪接位点下游的增强子。该增强子含有简单序列GGGGCUG的六个拷贝。增强子激活异源微外显子，并且当位于靶外显子的上游或下游时将起作用，这表明能够结合桥接剪接单元的因子。该序列的单个拷贝足以用于体内外显子包含，并且是已知的桥接哺乳动物剪接因子1（SF1）的结合位点。增强子及其结合的SF 1用于在外显子定义期间增加上游外显子的识别，使得与含有GGGGCUG重复序列的RNA的体外反应的竞争抑制上游内含子的剪接、剪接体在外显子的3'剪接位点上的组装以及SF 1的交联。这些结果表明一个模型，其中SF1桥的小外显子在初始组装，从而有效地扩展域的外显子。
Internal exon size in vertebrates occurs over a narrow size range. Experimentally, exons shorter than 50 nucleotides are poorly included in mRNA unless accompanied by strengthened splice sites or accessory sequences that act as splicing enhancers, suggesting steric interference between snRNPs and other splicing factors binding simultaneously to the 3' and 5' splice sites of microexons. Despite these problems, very small naturally occurring exons exist. Here we studied the factors and mechanism involved in recognizing a constitutively included six-nucleotide exon from the cardiac troponin T gene. Inclusion of this exon is dependent on an enhancer located downstream of the 5' splice site. This enhancer contains six copies of the simple sequence GGGGCUG. The enhancer activates heterologous microexons and will work when located either upstream or downstream of the target exon, suggesting an ability to bind factors that bridge splicing units. A single copy of this sequence is sufficient for in vivo exon inclusion and is the binding site for the known bridging mammalian splicing factor 1 (SF1). The enhancer and its bound SF1 act to increase recognition of the upstream exon during exon definition, such that competition of in vitro reactions with RNAs containing the GGGGCUG repeated sequence depress splicing of the upstream intron, assembly of the spliceosome on the 3' splice site of the exon, and cross-linking of SF1. These results suggest a model in which SF1 bridges the small exon during initial assembly, thereby effectively extending the domain of the exon.