Computational and Experimental Analysis of Vertebrate RNA Splicing

描述(申请人提供)：绝大多数人类基因需要RNA剪接才能表达，至少15%导致人类疾病的突变是通过破坏剪接来实现的。该项目的长期目标仍然是：了解RNA剪接特异性的基础-初级转录本中被RNA剪接机制识别并用于选择脊椎动物和其他生物剪接位点的序列的性质。这一长期目标集中在四个短期目标上：1)系统地鉴定可作为内含子剪接增强子(ISE)和沉默(ISS)元件的序列，完善我们对外显子剪接增强子(ESE)和沉默(ESS)元件的知识，并确定这些元件的上下文相关活性规则；2)确定对先前已确定的相当大比例的外显子剪接调控元件的剪接调控活性负责的反式作用因子；3)开发和应用高通量技术，以小鼠造血系统为模型，绘制发生在整个基因组发育过程中和对外部刺激做出反应的剪接异构体的表达变化；4)确定不同类别剪接调控元件之间的功能关系-可加性、亚可加性、协同性等，开发相关的评分系统，以改进模拟剪接和预测人类基因突变或多态的剪接表型的算法。在解决这些问题时，我们将使用计算方法与分子遗传学和功能基因组学方法的协同组合。了解剪接调控序列和蛋白质将有助于理解随着细胞增殖而发生的基因RNA版本(异构体)表达的变化，并可能识别特定的蛋白质或RNA靶点，用于治疗血液中的增殖性疾病，如白血病、淋巴瘤和自身免疫性疾病。准确模拟剪接的能力将使基因组注释得到改进，并将有助于识别与人类疾病相关的特定基因、突变和多态。

DESCRIPTION (provided by applicant): The vast majority of human genes require RNA splicing for their expression, and at least 15% of the mutations that cause human diseases do so by disrupting splicing. The long term objective of this project remains: to understand the basis of RNA splicing specificity - the nature of the sequences in primary transcripts which are recognized by the RNA splicing machinery and used in the selection of splice sites in vertebrates and other organisms. This long-term objective is focused on four shorter-term aims: 1) to systematically identify sequences that can act as intronic splicing enhancer (ISE) and silencer (ISS) elements, to refine our knowledge of exonic splicing enhancer (ESE) and silencer (ESS) elements, and to determine rules for the context-dependent activity of these elements; 2) to identify the trans-acting factors responsible for the splicing regulatory activity of a significant proportion of the exonic splicing regulatory elements identified previously; 3) to develop and apply high-throughput technologies to map changes in expression of spliced isoforms that occur genome-wide during development and in response to external stimuli, using the murine hematopoietic system as a model; 4) to determine functional relationships - additivity, sub-additivity, synergism, etc. - between different classes of splicing regulatory elements, to develop associated scoring systems to improve algorithms that simulate splicing and predict splicing phenotypes of mutations or polymorphisms in human genes. In addressing these questions, we will use a synergistic combination of computational methods with molecular genetic and functional genomic approaches. Knowledge of splicing regulatory sequences and proteins will aid in understanding the changes that occur in the expression of RNA versions (isoforms) of genes as cells proliferate, and may identify specific protein or RNA targets for therapuetic intervention in hyperproliferative diseases of the blood such as leukemias, lymphomas and autoimmune diseases. The ability to accurately simulate splicing will enable improved genome annotation and will facilitate identification of specific genes, mutations and polymorphisms associated with human diseases.