Mechanisms of Enhancer Dependent SpliceSite Activation

前体mRNA剪接是大多数后生动物基因表达所需的基本过程。缺陷 剪接导致许多人类遗传性疾病，并且剪接突变涉及生长的许多基因 控制已经涉及多种类型的癌症。了解前mRNA的基本机制 因此，剪接和剪接位点识别是理解调控基因表达的基础， 人类疾病虽然许多不同的顺式作用RNA剪接元件已被证明影响 选择性剪接，目前尚不清楚它们的组合贡献如何介导外显子包含或 排斥。使实验性破译选择性剪接决定的任务复杂化的是， 大多数人类基因含有多个内含子和外显子，其通常表现出比 简单地在两个竞争剪接位点之间选择。本次更新申请重点在于了解 以预测选择性剪接为长期目标的剪接位点选择调控机制 基于序列分析。下面概述的实验是建立在最令人兴奋的发现之上的。 在上一个融资期。历史上，SR蛋白与剪接激活有关， 而已知hnRNP抑制剪接反应。然而，新的全基因组分析 提示hnRNP样剪接因子也可以激活外显子包含。我们证明， 一类剪接调节因子具有促进或抑制剪接的能力， 取决于剪接调节子是结合在外显子内还是内含子内。因此，SR蛋白和 hnRNP在功能上是可互换的，它们对剪接的调节取决于它们的位置。 结合位点相对于剪接位点。剪接因子怎么可能激活或抑制 剪接体组装？我们建议进行补充实验，以确定 剪接调节蛋白从剪接激活子转换为剪接阻遏子的分子机制 （具体目标1和2）。在具体目标3中，我们将确定生活中的频率位置依赖性剪接 细胞，并使用新的分子见解来改善剪接预测。

Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. Defects in splicing lead to many human genetic diseases, and splicing mutations in a number of genes involved in growth control have been implicated in multiple types of cancer. Insights into the basic mechanisms of pre-mRNA splicing and splice site recognition are therefore fundamental to understanding regulated gene expression and human disease. While many different cis-acting RNA splicing elements have been shown to influence alternative splicing, it is currently unknown how their combinatorial contribution mediates exon inclusion or exclusion. Complicating the task of experimentally deciphering alternative splicing decisions is the fact that most human genes contain multiple introns and exons that often exhibit more complex splicing patterns than simply selecting between two competing splice sites. This renewal application focuses on understanding the mechanisms of regulated splice-site selection with the long-term goal to predict alternative splicing based on sequence analysis. The experiments outlined below build on the most exciting discoveries made during the previous funding period. Historically, SR proteins have been associated with splicing activation, whereas hnRNPs are known for their inhibition of the splicing reaction. However, new genome-wide analyses suggested that hnRNP-like splicing factors could also activate exon inclusion. We demonstrated that both classes of splicing regulators have the ability to promote or repress splicing, antagonistic activities that simply depend on whether the splicing regulator binds within the exon or within the intron. Thus, SR protein and hnRNPs are functionally interchangeable and their regulation of splicing is dependent on the location of their binding site relative to a splice site. How is it possible that a splicing factor can activate or repress spliceosome assembly? We propose to carry out complementing sets of experiments to determine the molecular mechanisms that switch splicing regulatory proteins from splicing activators to splicing repressors (Specific Aims 1 and 2). In Specific Aim 3 we will determine the frequency position-dependent splicing in living cells and use the new molecular insights to improve splicing predictions.