SAM68-induced regulation of cellular metabolism through mTOR mRNA splicing and stabilization

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Regulation of cell metabolism is critical for normal cell growth, homoeostasis, integrity and survival. It involves complex sequences of controlled biochemical reactions orchestrated by the mechanistic target of rapamycin (mTOR) signaling pathways. mTOR belongs to the phosphatidylinositol 3 -kinase- related protein family and is known to regulate cell growth, cell proliferation, cell motility, cell survival, protein synthesis and gene transcription. Hence, processes affecting either mTOR expression or its activity will have a major impact on cell metabolism.******In the initial phase of my NSERC research program, we identified SAM68 as a key regulator of mTOR splicing. We found that Sam68 depletion provoked an increase in intron 5 retention, creating a smaller mRNA termed mTORi5. While the exact molecular mechanism remains unknown, our recent investigations showed that SAM68 can interact with U1 snRNP, a core component of the spliceosome, which facilitates 5' splice site recognition. This suggests that interaction between SAM68 and spliceosome component might be essential for mTOR normal splicing and proper splice site recognition. Moreover, while mTORi5 mRNA can be detected, no protein product corresponding to this particular transcript was detected. Still, increased expression of the mTORi5 mRNA variant is associated with a drastic decrease in mTOR full-length protein expression, suggesting that Sam68 has the ability to regulate mTOR expression through a specific alternative splicing checkpoint of mTOR pre-mRNA.******Our hypothesis is that this SAM68--regulated splicing mechanism could be affecting mTOR mRNA stability, thus affecting its activity. Moreover, this mechanism could affect a broader range of mRNA and act as a general mechanism to regulate mRNA splicing and stability in a SAM68-dependent manner. This will be achieved by:******Aim 1: Determining the mechanism by which SAM68 can recruit key spliceosome components and regulates splicing mechanism.******Aim 2: Defining how SAM68 regulates mTOR mRNA stabilization and degradation.******Within this proposed research program, we will define a newly identified SAM68--regulated mechanism affecting alternative splicing and mRNA stability. While most of our research will be initiated using our mTOR model, we will also determine if this SAM68--regulated mechanism affects other mRNA that could act as novel translational safeguard mechanisms involved in cellular integrity.**