Real time measurements of splicing and probing spliceosome assembly models in living cells

活细胞中剪接和探测剪接体组装模型的实时测量

• 批准号: 135392659
• 负责人: Dr. Ute Schmidt
• 金额: --
• 依托单位: Institut de Génétique Moléculaire de Montpellier
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2010-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/135392659?language=en
• 关键词: Real; time; measurements; splicing; probing

Spliceosome assembly and splicing is a very dynamic and regulated process, but it is poorly under-stood in vivo. Further, many mRNA processing events, including capping, splicing, and 3'-end processing, occur at the transcription site. I propose to develop methods to visualize early RNA processing steps at the level of single genes using the MS2-reporter system. The reporters carry ms2-stem loops, which are specifically bound by the GFP-MS2 protein. When multiple copies are integrated into the genome of cells, the signal is amplified and mRNA processing factors become enriched at the mRNA transcription sites. Using both fixed and live cell techniques, I am able to: (1) obtain real-time measurements of splicing rates; (ii) measure residency times of splicing factors, thereby providing a kinetic framework of the spliceosomal assembly/disassembly cycle and (iii) probing plasticity and stability of defined spliceosomal cycles by blocking the spliceosome at specific steps. I will quantify different splicing steps and intron removal using either a wild-type reporter, which is spliced efficiently or mutant reporters, which are not spliced and block assembly at various steps. As several diseases are linked to mis-splicing or are a consequence of splice-site mutations, a spatiotemporal view on the splice reaction will be informative to develop strategies to correct for erroneous splicing in molecular therapies.

剪接体的组装和剪接是一个非常动态和受调控的过程，但在体内却知之甚少。此外，许多mRNA加工事件，包括盖帽、剪接和3'端加工，都发生在转录位点。我建议使用ms2报告系统开发方法，在单基因水平上可视化早期RNA加工步骤。报告基因携带ms2-茎环，它与GFP-MS2蛋白特异性结合。当多个拷贝被整合到细胞基因组中时，信号被放大，mRNA加工因子在mRNA转录位点变得丰富。使用固定细胞和活细胞技术，我能够：(1)获得剪接率的实时测量；（ii）测量剪接因子的停留时间，从而提供剪接体组装/拆卸周期的动力学框架；（iii）通过在特定步骤阻断剪接体来探测已定义剪接体周期的可塑性和稳定性。我将量化不同的剪接步骤和内含子的去除，使用野生型报告子（有效剪接）或突变报告子（未剪接），并在不同步骤阻断组装。由于一些疾病与剪接错误有关，或者是剪接位点突变的结果，因此对剪接反应的时空视图将为制定分子治疗中纠正错误剪接的策略提供信息。