Alternative splicing (AS) is an important post-transcriptional process regulating gene expression in the majority of intron-containing genes in eukaryotes. However, AS does not merely increase the coding potential of an organism, but also has a substantial influence on the regulation of gene expression, even more so under stress conditions or during developmental phases. In order to gain an even higher regulatory complexity with the aim to reach even finer tuning of gene expression, AS is coupled to a cytoplasmic RNA degradation pathway termed Nonsense-mediated decay (NMD). During the first round of translation, this mechanism recognizes a number of signals intrinsic to transcripts and efficiently degrades them. One of these signals is the presence of a premature termination codon (PTC) with a certain distance to the next exon junction (>50 nucleotides) which has been shown to inevitably trigger NMD. However, recent studies found that some of the PTC-positive transcripts, which are expected to be degraded by NMD, do not accumulate in NMD-defficient mutants. The aim of this study was to investigate the fate of such transcripts in living cells. Up to this point it was unknown, how such transcripts escape the NMD machinery; however, one hypothesis was that they may not be exported to the cytoplasm.
In order to investigate the subcellular localization of particular RNA transcript variants as well as their single molecule dynamics, it was necessary to establish a quantitative imaging approach guaranteeing robust statistical analysis. Speciffic splice variants were targeted by hybridization-sensitive probes (Molecular Beacon, MB) and imaged via standard confocal laser scanning microscopy. The system was first tested on protoplasts of a gene inducible A. thaliana culture and later adapted to monitor the subcellular distribution of splice variants in wild type cells. With this system, it was eventually possible to demonstrate that the investigated PTC-positive, but NMD-insensitive transcripts are retained within the nucleus, and thus escaped the NMD machinery. Also, via bulk fluorescence recovery after photobleaching (FRAP) experiments, it became clear that fully-spliced transcripts move faster throughout the nucleoplasm than their alternatively spliced intron-retaining counterparts. Additionally, single-molecule tracing experiments of MB-labelled mRNA splice variants were performed, resulting in the detection of individual mRNAs in living cells.
In conclusion, these findings contribute to the understanding of the dynamic behavior of individual mRNA splice variants in living plant cells, and solved a multitude of technical challenges arising from the in vivo quantitative imaging approach.
The opportunity for an independent project arose during the last year of my PhD-study and involved the implementation of a software tool for the analysis of terminal restriction fragment (TRF) southern blot data.
Also, the software tool for telomere length analysis is an important asset to the respective scientific community and is, for that reason, implemented as open source and published as open access literature.