Comprehensive Analysis of RAS-mediated Alternative Splicing

Alternative splicing is a mechanism for generating functionally different proteins from one gene resulting in increase in protein diversity. However, our knowledge of molecular mechanisms of alternative splicing regulated by intracellular signals is extremely limited, because there was no effective way to detect alternative splicing comprehensively and quantitatively. Recently, RNA sequencing method enables us to quantitate expression level of each splicing variant comprehensively. In this study, we focused the effect on alternative splicing mediated by the RAS-MAPK signaling pathway, which is well known as an oncogenic pathway. We examined the function of the RAS-MAPK signal by RNA sequencing method.

We overexpressed constitutive-active form of RAS (RAS^G12V) in mouse embryonic fibroblast cell line, NIH3T3, to activate the RAS-MAPK pathway, extracted whole mRNAs from RAS-activated cells and control cells, and sequenced cDNA using next-generation sequencer, Illumina GAIIx. The RNA sequence tags were mapped to the mouse genome. By the number of tags mapped to each exon, we quantitated the expression level of each splicing variant to find out the genes, whose splicing patterns were altered by RAS-MAPK signal. To validate the results of RNA sequencing and examine the impacts on protein expression, we carried out quantitative PCR and Western blotting on several gene products.

These analyses revealed that the splicing patterns of some genes were affected by the RAS-MAPK signaling, including H2AFY, which codes the atypical histone variant macroH2A1. We found that these splicing changes, validated by qPCR, induced the expression of different proteins.

It is known that macroH2A1 is related to expression of wide-ranged genes. Moreover, it is reported that there are different alternative splicing patterns of macroH2A1 between highly proliferative cells such as cancer cells and normal cells, suggesting that the splicing pattern of macroH2A1 regulates cell proliferation. Our findings suggest that activation of the RAS-MAPK signal might contribute to oncogenesis by alteration of the alternative splicing pattern. It emphasizes the importance of paying attention not only to the amount of transcript but also to the change of alternative splicing patterns, when we interpret the biological significance of RNA expression.