SPLICING OF HUMAN MRNA IN VITRO

人类 mRNA 的体外剪接

• 批准号: 2176815
• 负责人: RYSZARD KOLE
• 金额: $ 18.61万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-12-01 至 1995-12-11
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176815
• 关键词: Adenoviridae; HeLa cells; RNA splicing; RNase protection assay; cell free system; gel electrophoresis; gene expression; genetic transcription; globin; human genetic material tag; human tissue; messenger RNA; nucleic acid sequence; precursor mRNA; radiotracer; tissue /cell culture; transfection

The long-term goal of this project, which remains unchanged from the previous granting periods, is the study of pre-mRNA splicing in human cells.The main focus of this proposal will be the study of sequence elements in pre-mRNA that play a role in the selection of splice sites in a multiintron precursor. The mechanisms that determine splice site selection in constitutive and alternative splicing are not well understood and present one of the major unresolved questions in pre-mRNA splicing. This proposal will attempt to elucidate these mechanisms by the following experimental approaches: 1) Transcripts with a short internal exon and two introns, a model for a multiintron pre-mRNA, will be spliced in vitro. Preliminary results show that in such model transcripts the internal exon is either skipped or included in the spliced product depending on the sequence of the splice sites and the length of the internal exon. The details of the mechanism of splice site selection as exemplified by exon skipping/inclusion will be further elucidated. In particular, a hypothesis will be tested that the selection of splice sites depends on the interplay between the "strength" of the splice sites flanking the short internal exon and the length of this exon. Additional mechanisms of splice site selection such as "exon definition" and splice site competition win also be investigated. 2) The interactions of splicing factors with model pre-mRNAs mentioned above will be investigated. Formation of splicing complexes will be analyzed by native gel electrophoresis. The kinetics and nature of the interactions of splicing factors with splice sites will be determined by nuclease protection assays. The model transcripts will also be used to study the role of splicing factors in alternative splicing. A hypothesis will be tested that the relative concentrations of "housekeeping" splicing factors are responsible for alternative exon skipping or exon inclusion. We will also test whether factors that appear to be specific for certain alternatively spliced pre-mRNAs can induce alternative splicing in our model systems. 3) The patterns of splicing of model pre-mRNAs in vitro, in cell-free extracts, and in vivo in transiently and stably transfected cells will be compared. The goal of these experiments is to determine if coupling of transcription and splicing affects the patterns of splice site selection and if there are any additional mechanisms that control splice site selection in vivo.

该项目的长期目标，与该项目保持不变 以前的授予期是对人类的前mRNA剪接的研究 单元格。该提案的主要重点将是序列的研究 前MRNA中的元素在选择剪接站点中发挥作用 多动龙前体。 确定剪接位点的机制 在本构和替代剪接中的选择不好 理解并提出了前MRNA中主要未解决的问题之一 剪接。 该建议将尝试通过 以下实验方法：1）简短的转录本 内外外显子和两个内含子，一个多动龙前mRNA的模型，将 在体外被剪接。初步结果表明，在这种模型中 成绩单内部外显子被跳过或包含在 剪接的产品取决于剪接位点的序列和 内外外显子的长度。 剪接网站机制的细节 外显子跳过/包含的选择将进一步 阐明。 特别是，将检验一个假设 剪接站点的选择取决于“强度”之间的相互作用 剪接位点的侧面外显子和长度的侧面 这个外显子。 剪接站点选择的其他机制，例如“外显子 定义“和剪接场地竞赛也将赢得胜利。2） 剪接因子与上面提到的模型PREMNA的相互作用 将被调查。 将分析剪接复合物的形成 通过天然凝胶电泳。 动力学和性质 剪接因子与剪接位点的相互作用将由 核酸酶保护测定法。 模型成绩单也将用于 研究剪接因子在替代剪接中的作用。 一个假设 将测试“客房管理”的相对浓度 剪接因子负责替代外显子跳过或外显子 包容。 我们还将测试似乎是特定的因素 对于某些剪接的pre-mRNA可以诱导替代 在我们的模型系统中进行拼接。 3）模型的剪接模式 体外MRNA，无细胞提取物以及瞬时和体内的MRNA 将比较稳定的转染细胞。 这些实验的目标 是确定转录和剪接的耦合是否影响 剪接网站选择的模式，以及是否还有其他 在体内控制剪接位点选择的机制。