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

剪接体的组装和剪接是一个非常动态和受调控的过程，但在体内对其了解很少。此外，许多mRNA加工事件，包括封顶、剪接和3‘端加工，都发生在转录位点。我建议开发使用MS2报告系统在单基因水平上可视化早期RNA加工步骤的方法。这些记者携带MS2-茎环，它特定地与GFP-MS2蛋白结合。当多个拷贝整合到细胞基因组中时，信号被放大，信使核糖核酸的处理因子在信使核糖核酸转录位点变得丰富。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；以及(Iii)通过在特定步骤阻断剪接体来探测定义的剪接体周期的可塑性和稳定性。我将使用野生型记者和突变型记者来量化不同的剪接步骤和内含子的去除，野生型记者是高效剪接的，突变型记者是不剪接的，并在不同的步骤上阻止组装。由于一些疾病与错误剪接有关，或者是剪接位点突变的结果，剪接反应的时空观点将有助于制定策略，纠正分子治疗中的错误剪接。

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.