Single cell alternative splicing

JUAN PABLO FEDEDA; ALEJANDRO COLMAN-LERNER; ALBERTO KORNBLIHTT

Bariloche, Pcia de Río Negro

Congreso; Cell Biology, Signaling and Alternative Splicing; 2007

nterdisciplinary Focus Meeting of the European Alternative Splicing Network (EURASNET)

Our information on alternative splicing derives from the average behaviour of populations of cells from cultures and tissues. Despite the huge progress in our understanding of this process, several questions remain. For example, we do not yet know about the splicing behaviour of single cells, the ultimate biological units. Is there a large cell to cell variation at the alternative processing level in a population of genetically clonal cells, or most cells behave like the population mean? How do different individual cells respond to stimuli that we and others have shown to change the average alternative splicing? If differences exist in alternative splicing between cells of the same clonal population, are these differences inherited in a cell lineage? To address these questions, we developed a two-colour fluorescent protein-based alternative splicing reporter in which the human fibronectin EDI exon is the alternatively spliced region. When the EDI exon is included, the resulting fusion polypeptide encodes the yellow fluorescent protein. On the contrary, when EDI is skipped, the cyan fluorescent protein is translated.  Preliminary microscopy results show a good correlation between this newly developed fluorescent protein splicing reporter and our well characterized RT-PCR method to estimate EDI inclusion ratios. We also observed a similar change in inclusion ratio when we used reporters bearing mutations that force the inclusion or skipping of the alternative exon. In experiments where cells were allowed to proliferate for 48 hours, there was a significant bias to find cells with similar alternative splicing ratios grouped together, suggesting that the splicing ratio is inherited and therefore that the cell to cell variation observed is not the result of stochastic processes.