Splicing Factor SF2/ASF at the crossroad between cell proliferation, differentiation and the DNA damage response.

Splicing factor SF2/ASF plays a crucial role in several aspect of RNA metabolism, from splicing decisions to mRNA export, translation and RNA stability. We have recently shown that during in vitro EMT (epithelial to mesenchymal cell transition) SF2/ASF expression is controlled at the post-transcriptional level through alternative splicing of the intron in the 3'UTR, which promotes degradation of SF2/ASF transcripts via the NMD pathway. Key elements in this regulatory circuit are: 1) soluble factors expressed by epithelial cells, 2) the ERK1/2 kinase and 3) the phosphorylation status of the splicing regulator Sam68. Our in vitro model of EMT in colon adenocarcinoma SW480 cells simply depends on cell density: low-density, proliferating SW480 cells display morphological and molecular markers of fibroblast-like mesenchymal cells, whereas high-density cells cease to proliferate and show a typical cobblestone epithelial phenotype. Interestingly, the same modulation of the splicing profile of SF2/ASF transcripts in response to cell density takes place even in cells that are epithelial both a low and high density such as colon adenocarcinoma HT-29 and gastric carcinoma T47D cells. Notably, in all these cell lines, the splicing profile in the 3'UTR of SF2/ASF transcripts is controlled by the signaling pathway identified by the ERK kinase. It is conceivable that the higher expression of SF2/ASF in low-density cells is necessary for cell proliferation and for the movement of mesenchymal cells. More recently, we have found that the expression and activity of SF2/ASF are controlled also in response to DNA damage. This was initially observed in 46BR.1G1 cells, which derive from a patient with a defect of DNA ligase I (LigI), a key enzyme in DNA replication. LigI deficiency results in accumulation of both single- and double-stranded DNA breaks behind the replication fork. The 46BR.1G1 defect is efficiently corrected by over-expressing the wild-type enzyme in 7A3 cells. Comparative analysis of the proteome complexity identified a number of RNA processing factors, among which SF2/ASF, with different expression in 46BR.1G1 and 7A3 cells. Interestingly, the phosphorylation profile of SF2/ASF and splicing of the intron in the 3'UTR are altered in 46BR1G1 cells compared to 7A3 and control fibroblasts. The same alterations occur in normal fibroblasts after treatment with DNA damaging agents. These changes of phosphorylation pattern and expression level are accompanied by modifications in the splicing profile of pre-mRNAs that are known substrates of SF2/ASF. Thus, different stimuli such as cell density and DNA damage, both of which relevant for tumor progression, may impact on the level and activity SF2/ASF.