Exon recognition during constitutive pre-mRNA splicing

描述(申请人提供)：我们DNA中固有的遗传信息的表达包括4个基本过程：1)转录成RNA，2)将RNA中的信息片段拼接成信使RNA，或信使RNA，3)将信使RNA翻译成蛋白质，4)修饰蛋白质使其有效。这项建议侧重于第二个过程，即前mRNA剪接。虽然剪接反应的化学成分已经很好地了解了，但细胞如何识别编码蛋白质的少数相对较短的前mRNA区域(外显子，~100个核苷酸(NT)长，每个转录本~10个)的界限尚不清楚。剪接位点本身由具有特定特征的序列组成。例如，每个剪接出的区域(内含子)几乎总是以GT开始，以AG序列结束。然而，剪接位点序列不够独特，不能提供明确的标记。为了破译“剪接密码”，我们将采用4种方法，即允许识别位于前mRNA或DNA序列中的剪接位点的序列元素和规则：1)我们将把所有可能的6个核苷酸(4096)的序列添加到一个弱化的外显子中，以定义那些可以增强剪接的完整列表。通过重复这些实验并比较以不同方式改变外显子后发现的序列，我们将了解整个序列的不同部分是如何相互作用来产生信号的。这些实验利用了最近开发的大量DNA短区域测序的方法。2)我们发现，外显子外的有限内含子区域可以在剪接位点识别中发挥强大的作用，但对这些序列的一般性质或作用知之甚少。我们将研究这些内含子增强子的位置和蛋白质结合特性对剪接和染色质结构的影响。我们将细胞基因用于这一目的，是对目前使用的不那么自然的测试系统的改进。3)现在看来，影响剪接的信号密度非常高，因此对自然序列的任何操作都可能同时改变不止一个信号。为了将这种影响降到最低，我们将构建使用已知效果的绝缘模块(剪接的增强剂和消音器)设计的合成外显子。通过将这些模块放在不同的排列中，我们将学习管理它们相互作用的规则。4)对人类基因组序列的统计分析成功地预测了外显子增强子和消音子。我们将扩展这种计算方法来寻找内含子和外显子信号，这些内含子和外显子信号可以协同增强剪接，并可能起到沉默错误剪接位点的作用。许多人类遗传病是由剪接缺陷引起的，癌细胞经常表现出异常的剪接模式。对剪接密码的了解将使这一过程能够针对治疗用途，如纠正遗传疾病中的缺陷，或扰乱肿瘤中的有害剪接事件。 公共卫生相关性： 人类基因通过将它们的信息转化为操作我们细胞的蛋白质来控制我们的生活。这种遗传信息以片段的形式存在，必须拼接在一起才有意义，拼接过程的中断会导致许多遗传病，并可能导致癌症。我们的建议旨在了解这种拼接是如何发生的。

DESCRIPTION (provided by applicant): The expression of the genetic information inherent in our DNA includes 4 basic processes: 1) transcription into RNA, 2) splicing together the fragments of information in this RNA into messenger RNA, or mRNA ; 3) translation of the mRNA into proteins; 4) modifying the proteins to make them effective. This proposal focuses on the second process, pre-mRNA splicing. Although the chemistry of the splicing reaction is fairly well understood, it is not yet clear as how the cell recognizes the demarcation of the few relatively short regions of the pre-mRNA that code for protein (the exons, ~100 nucleotides (nt) long, ~10 per transcript) within a long (~20,000 nt) pre-mRNA molecule. The splice sites themselves are comprised of sequences with specific features. For example, each spliced out region (the intron) almost always starts with a GT and ends with an AG sequence. However, the splice site sequences are not distinctive enough to provide an unambiguous mark. We will pursue 4 approaches with the aim of deciphering the "splicing code," i.e., the sequence elements and rules that allow recognition of splice sites lying within the sequence of the pre-mRNA or DNA: 1) We will add all possible sequences of 6 nt (4096) into a weakened exon to define the complete list of those that can enhance splicing. By repeating this experiments and comparing the sequences found after altering the exon in various ways, we will learn how different parts of the overall sequence interact to create a signal. These experiments exploit recently developed methods for massive sequencing of short regions of DNA. 2) We have found that limited intronic regions just outside the exon can play powerful roles in splice site recognition but little is known about the general nature or action of these sequences. We will investigate the effect of the position and protein-binding properties of these intronic enhancers on splicing and on chromatin structure. Our use of a cellular gene for this purpose is an improvement over less natural test systems currently in use. 3) It now appears that the density of signals influencing splicing is very high, so that any manipulation of a natural sequence is likely to change more than one signal at once. To minimize this effect we will build synthetic exons designed using insulated modules of known effect (enhancers and silencers of splicing). By placing these modules in various permutations, we will learn the rules governing their interactions. 4) Statistical analysis of the human genome sequence has allowed the successful prediction of exonic enhancers and silencers. We will extend such computational approaches to search for intronic and exonic signals that cooperate to enhance splicing and that may act to silence false splice sites. Many human genetic diseases are caused by splicing deficiencies and cancer cells often exhibit abnormal splicing patterns. A knowledge of the splicing code will enable this process to be targeted for therapeutic use, such as correcting a deficiency in a genetic disease, or disrupting a harmful splicing event in a tumor. PUBLIC HEALTH RELEVANCE: Human genes control our lives by having their information translated into the proteins that operate our cells. That genetic information is present as fragments that must be spliced together to make any sense, and disruption of the splicing process causes many genetic diseases and can contribute to cancer. Our proposal is aimed at understanding how this splicing takes place.