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

点击翻译按钮获取中文摘要

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.