Alternative splicing is responsible for much of the transcriptomic and proteomic diversity observed in eukaryotes and involves combinatorial regulation by many cis-acting elements and trans-acting factors. SR and hnRNP splicing regulatory proteins often have opposing effects on splicing efficiency depending on where they bind the pre-mRNA relative to the splice site. Position-dependent splicing repression occurs at spliceosomal E-complex, suggesting that U1 snRNP binds but cannot facilitate higher order spliceosomal assembly. To test the hypothesis that the structure of U1 snRNA changes during activation or repression, we developed a method to structure-probe native U1 snRNP in enriched conformations that mimic activated or repressed spliceosomal E-complexes. While the core of U1 snRNA is highly structured, the 5' end of U1 snRNA shows different SHAPE reactivities and psoralen crosslinking efficiencies depending on where splicing regulatory elements are located relative to the 5' splice site. A motif within the 5' splice site binding region of U1 snRNA is more reactive towards SHAPE electrophiles when repressors are bound, suggesting U1 snRNA is bound, but less base paired. These observations demonstrate that splicing regulators modulate splice site selection allosterically.

中文翻译：

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通过剪接调节蛋白控制剪接体组装对 U1 snRNP 的变构调节

在真核生物中观察到的转录组学和蛋白质组学多样性主要由可变剪接负责，并且涉及许多顺式作用元件和反式作用因子的组合调节。SR 和 hnRNP 剪接调节蛋白通常对剪接效率有相反的影响，这取决于它们相对于剪接位点结合前体 mRNA 的位置。位置依赖性剪接抑制发生在剪接体 E 复合体，表明 U1 snRNP 结合但不能促进更高阶的剪接体组装。为了测试 U1 snRNA 的结构在激活或抑制过程中发生变化的假设，我们开发了一种方法来构建探测天然 U1 snRNP 的富集构象，模拟激活或抑制的剪接体 E 复合物。虽然 U1 snRNA 的核心是高度结构化的，但 5' U1 snRNA 的末端显示不同的 SHAPE 反应性和补骨脂素交联效率，这取决于剪接调节元件相对于 5' 剪接位点的位置。当阻遏物被结合时，U1 snRNA 的 5' 剪接位点结合区域内的一个基序对 SHAPE 亲电试剂更具反应性，表明 U1 snRNA 被结合，但碱基配对较少。这些观察结果表明剪接调节剂以变构方式调节剪接位点选择。