Singh R, Banerjee H, Green M R
Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, 80309, USA.
RNA. 2000 Jun;6(6):901-11. doi: 10.1017/s1355838200000376.
The polypyrimidine-tract (Py-tract) adjacent to 3' splice sites is an essential splicing signal and is recognized by several proteins, including the general splicing factor U2AF65 and the highly specific splicing repressor Sex-lethal (SXL). They both contain ribonucleoprotein-consensus RNA-binding motifs. However, U2AF65 recognizes a wide variety of Py-tracts, whereas SXL recognizes specific Py-tracts such as the nonsex-specific Py-tract of the transformer pre-mRNA. It is not understood how these seemingly similar proteins differentially recognize the Py-tract. To define these interactions, we used chemical interference and protection assays, saturation mutagenesis, and RNAs containing modified nucleotides. We find that these proteins recognize distinct features of the RNA. First, although uracils within the Py-tract are protected from chemical modification by both of these proteins, modification of any one of seven uracils by hydrazine, or any of eight phosphates by ethylnitrosourea strongly interfered with the binding of SXL only. Second, the 2' hydroxyl groups or backbone conformation appeared important for the binding of SXL, but not U2AF65. Third, although any of the bases (cytosine >> adenine > guanine) could substitute for uracils for U2AF65 binding, only guanine partially substituted for certain uracils for SXL binding. The different dependence on individual contacts and nucleotide preference may provide a basis for the different RNA-binding specificities and thus functions of U2AF65 and SXL in 3' splice site choice.
与3'剪接位点相邻的多嘧啶序列（Py序列）是一种重要的剪接信号，可被多种蛋白质识别，包括一般剪接因子U2AF65和高度特异性的剪接抑制因子性致死蛋白（SXL）。它们都含有核糖核蛋白共有RNA结合基序。然而，U2AF65能识别多种Py序列，而SXL只识别特定的Py序列，如transformer前体mRNA的非性别特异性Py序列。目前尚不清楚这些看似相似的蛋白质如何差异识别Py序列。为了确定这些相互作用，我们使用了化学干扰和保护试验、饱和诱变以及含有修饰核苷酸的RNA。我们发现这些蛋白质识别RNA的不同特征。首先，尽管Py序列中的尿嘧啶受到这两种蛋白质的保护而免受化学修饰，但用肼对七个尿嘧啶中的任何一个进行修饰，或用乙基亚硝基脲对八个磷酸中的任何一个进行修饰，只会强烈干扰SXL的结合。其次，2'羟基或主链构象对SXL的结合似乎很重要，但对U2AF65的结合不重要。第三，尽管任何碱基（胞嘧啶>>腺嘌呤>鸟嘌呤）都可以替代尿嘧啶用于U2AF65的结合，但只有鸟嘌呤部分替代某些尿嘧啶用于SXL的结合。对单个接触和核苷酸偏好的不同依赖性可能为U2AF65和SXL在3'剪接位点选择中不同的RNA结合特异性及功能提供基础。
