A novel approach to describe a U1 snRNA binding site
A novel approach to describe a U1 snRNA binding site
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DOI：
10.1093/nar/gkg901
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发表时间：
2003-12-01
影响因子：
14.9
通讯作者：
Schaal, H
中科院分区：
文献类型：
--
作者：
Freund, M;Asang, C;Schaal, H
U1 snRNA 和剪接供体 (SD) 位点之间形成的 RNA 双链体可以保护前 mRNA 在剪接前免遭降解，并启动剪接体的形成。使用亚基因组 HIV-1 表达载体，通过糖蛋白 env 的表达分析来监测这一过程，糖蛋白 env 的形成关键取决于功能性 SD4。我们系统地推导了一个氢键模型，用于描述 U1 snRNA 的游离 5' 端和 5' 剪接位点之间的互补性以及用 5' 剪接位点突变载体瞬时转染 HeLa-T4(+) 细胞后的众多突变。由此产生的模型考虑了跨越外显子的三个最远 3' 碱基对（-3 至 -1）和内含子最远的 8 个碱基对（+1 至 +8）的区域中预测的氢键形成的数量、相互依赖性和邻域关系。该模型由对 U1 snRNA 结合位点进行分类的算法表示，这些结合位点可以或不能在 Rev 介导的 env 表达方面功能上替代 SD4。在人类 ATM 基因 5' 剪接位点突变的数据集中，我们发现算法分类和外显子跳跃之间存在显着相关性（P = 0.018，chi(2) 检验），表明该模型的适用性远远超出了 HIV-1 剪接。然而，算法分类不能被视为 SD 使用的绝对度量，因为它可能会被上游序列元素修改。在 SD4 的上游，我们鉴定了一个支持 ASF/SF2 结合的片段。该位点内突变的 GAR 核苷酸重复减少了 SD4 依赖性 Rev 介导的 env 表达，这可以通过人为增加 SD4 的互补性来平衡。
RNA duplex formation between U1 snRNA and a splice donor (SD) site can protect pre-mRNA from degradation prior to splicing and initiates formation of the spliceosome. This process was monitored, using sub-genomic HIV-1 expression vectors, by expression analysis of the glycoprotein env, whose formation critically depends on functional SD4. We systematically derived a hydrogen bond model for the complementarity between the free 5' end of U1 snRNA and 5' splice sites and numerous mutations following transient transfection of HeLa-T4(+) cells with 5' splice site mutated vectors. The resulting model takes into account number, interdependence and neighborhood relationships of predicted hydrogen bond formation in a region spanning the three most 3' base pairs of the exon (-3 to -1) and the eight most 5' base pairs of the intron (+1 to +8). The model is represented by an algorithm classifying U1 snRNA binding sites which can or cannot functionally substitute SD4 with respect to Rev-mediated env expression. In a data set of 5' splice site mutations of the human ATM gene we found a significant correlation between the algorithmic classification and exon skipping (P = 0.018, chi(2)-test), showing that the applicability of the proposed model reaches far beyond HIV-1 splicing. However, the algorithmic classification must not be taken as an absolute measure of SD usage as it may be modified by upstream sequence elements. Upstream to SD4 we identified a fragment supporting ASF/SF2 binding. Mutating GAR nucleotide repeats within this site decreased the SD4-dependent Rev-mediated env expression, which could be balanced simply by artificially increasing the complementarity of SD4.