Real time measurements of splicing and probing spliceosome assembly models in living cells
Zusammenfassung der Projektergebnisse
As gene expression steps such as transcription, pre‐mRNA processing, and nucleo‐cytoplasmic export often occur in specialized subcellular compartments that carry specific functions, it is important to study them in their native environment. In the past, I focussed on the spatio‐temporal formation of mRNP particles in the nucleus and in particular on splicing and post‐splicing events. Splicing is at the heart of gene expression because it increases the coding capacity of our genome, but also it functions in stimulating transcription, 3' end processing, nucleo‐cytoplamic transport and quality control. The fellowship of the German Research Foundation (DFG) was used to work on splicing kinetics in real time and in live cells. However, in the course of my studies, I made another important discovery on the link between splicing and nuclear retention that motivated further projects which are currently in preparation. In detail, my most important results are: 1.) Splicing kinetics mimic a pseudo‐deterministic process. Using FRAP and MS2‐tagged introns, I was the first to analyze the kinetics of splicing in live‐cell and real‐time. I found that intron removal occurs in minutes and is best described by a model where several successive steps are rate limiting. This is important, as each single pre‐mRNA molecule is predicted to require a similar time to splice, and this greatly improves the regulation of alternative splicing. 2.) Unspliceable pre‐mRNAs are subjected to a quality‐control deadenylation mechanism. Using a combination of biochemical and imaging techniques, I observed that pre‐mRNA that assemble spliceosomes but that cannot splice are retained in the nucleus, at their transcription site, and I showed that they are deadenylated. This novel quality‐control step of the splicing reaction is important, as it prevents translation of incompletely spliced pre‐mRNAs, potentially producing toxic proteins. It is complementary to NMD as this process deals only with spliced mRNAs in mammals. 3.) Transcription initiation sites control splicing. Using reporter constructs and systematic sequencing of the mRNAs 5' and 3' ends, I discovered that a fraction of the reporter RNAs initiate transcription at ‐4, and that these RNAs are not competent for splicing. It is the first time that transcription initiation sites are shown to control splicing, and this new regulatory link has important consequences for gene expression. Points 2 and 3 were unexpected discoveries that I explored further.
Projektbezogene Publikationen (Auswahl)
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Real‐time imaging of co‐transcriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation. J Cell Biol. 2011 May 30;193(5):819‐29
Ute Schmidt, Basyuk E, Robert MC, Yoshida M, Villemin JP, Auboeuf D, Aitken S, Bertrand E
