Earlier we have showed that tumor associated

Earlier we have showed that tumor associated sFas mRNA isoform profile and splicing factors expression levels in solid tumors do not correspond to profiles that are observed in cell lines [45]. In the present study, for the first time we show that Fas but not Rac1 pre-mRNA's alternative splicing is regulated by cellular microenvironment, such as hypoxia. Also we demonstrate that splicing factors hnRNPA1 and SPF45, shown to be involved in Fas pre-mRNA alternative splicing regulation in normoxic cells, do not influence splice site choice under hypoxic conditions.
Materials and methods
Results
Discussion Splicing is tightly regulated in different tissues and developmental stages. Splicing machinery heavily contributes to the ability of cells to adapt to different developmental stages and altered cellular conditions. Hypoxia is an important regulator of physiologic processes, including erythropoiesis, angiogenesis and glycolysis [51]. Decreased oxygen levels within a cell result in HIF induction and the subsequent transcription of a number of target genes that promote cgrp antagonist to this environmental stress. The list of HIF targets includes genes affecting metabolism, oxygen delivery, angiogenesis and cellular survival [52]. Currently more and more data shows changes in cellular processes due to changes in cellular microenvironment [[53], [54], [55], [56]]. Besides pathological hypoxic conditions (e.g., inflammation, solid tumor formation, lung disease, or myocardial infarction), physiological hypoxia can also occur among healthy individuals during embryonic development or prolonged stay at high altitude [57,58]. Pre-mRNA splicing is an important process for cellular adaptation to microenvironmental changes [15]. It was shown that Fas mRNA isoform formation profile in tumors does not correspond to observe mRNA formation profiles in tumor cell lines, derived from the same organs. Fas tumor associated mRNA isoform levels in cancer cell lines are more similar to the mRNA isoform profiles observed in healthy tissues rather than in tumors [45]. Our performed studies showed that Fas but not Rac1b alternative pre-mRNA splicing is regulated by hypoxia, i.e. by changed cellular microenvironment. As hypoxia does not influence Fas pre-mRNAs cellular expression levels (Fig. 1B), we conclude that increase in sFas mRNA isoform expression in hypoxic cells is due to changes in pre-mRNA splicing. Hypoxia is a usual cellular microenvironment in solid tumors [5,59] thus it is not surprising that elevated production of soluble Fas (sFas) has been observed in a wide range of cancers. Our identified hypoxia-dependent increase in sFas mRNA formation in several cell lines offers the explanation how alternative splicing contributes to the cells’ ability to overcome apoptosis and helps cells to survive under reduced oxygen tension. Several individual factors (hnRNPA1, SPF45, TIA1/TIAR, PTB, hnRNPC, RBM10) have been identified as regulators of alternative Fas pre-mRNA splicing, particularly splicing of exon 6, in normoxic cells [27,31,33,[60], [61], [62], [63]]. Two of them, SPF45 and hnRNPA1, are well described as regulators possessing opposite regulating features on Fas pre-mRNA alternative splicing [34,64] therefore they were chosen for Fas alternative hypoxia-dependent splicing regulation studies. Splicing factors hnRNPA1 and SPF45 individually can regulate alternative Fas pre-mRNA splicing in a dose dependent manner in normoxic cells [28,29,34]. We were expecting to find a correlation between expression of these two factors: in cells cultivated under hypoxic and/or normoxic conditions. However, our results revealed that hypoxic conditions do not change hnRNPA1 and SPF45 protein expression levels in all four tested cell lines and we could not find any evidence that these factors could be involved in Fas alternative splicing regulation in a dose dependent manner in hypoxic cells. Also our data revealed that reduction of cellular expression of these factors did not significantly influence Fas alternative pre-mRNA splicing. Only the reduction of hnRNPA1 cellular levels slightly promotes sFas mRNA isoform formation in normoxic but not in hypoxic cells. However, our observation indicates that these changes are not essential for alternative Fas pre-mRNA splicing in hypoxic cells. Such contradiction with published results, observed in normoxic cells, could be explained by the fact, that in our studies we have used cellular endogenous RNA while in all published studies Fas minigene was used [28,29,34]. Also it was shown that in hypoxic cells SR proteins are hyperphosphorylated as compared to cells cultivated under normoxic conditions [15]. This finding suggests that splicing factors activity in cells cultured under hypoxic conditions are different from their activity observed in cells cultured under normoxic conditions. That also could explain observed differences in SPF45 and hnRNPA1 role in hypoxia dependent Fas alternative splicing regulation as compared to normoxic cells. It is also not possible to exclude that other factors might play an essential role in hypoxia-dependent Fas alternative splicing (particularly exon 6 skipping) regulation in hypoxic cells.