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

细胞代谢的调节对细胞的正常生长、动态平衡、完整性和生存至关重要。它涉及由雷帕霉素靶标(MTOR)信号通路编排的复杂的受控生化反应序列。MTOR属于磷脂酰肌醇3-激酶相关蛋白家族，调节细胞生长、细胞增殖、细胞运动、细胞存活、蛋白质合成和基因转录。因此，影响mTOR表达或其活性的过程将对细胞代谢产生重大影响。 在我的NSERC研究计划的最初阶段，我们发现SAM68是mTOR剪接的关键调控因子。我们发现Sam68缺失引起内含子5保留增加，产生了一个更小的名为mTORi5的mRNA。虽然确切的分子机制尚不清楚，但我们最近的研究表明，SAM68可以与剪接体的核心成分U1 SnRNP相互作用，从而促进5‘剪接位点的识别。这提示SAM68和剪接体成分之间的相互作用可能是mTOR正常剪接和正确的剪接位点识别所必需的。此外，虽然可以检测到mTORi5mRNA，但没有检测到与该特定转录物相对应的蛋白质产物。尽管如此，mTORi5mRNA变体的表达增加与mTOR全长蛋白表达的急剧下降有关，这表明Sam68有能力通过mTOR前-mRNA的特定选择性剪接检查点来调节mTOR的表达。 我们的假设是，这种SAM68调控的剪接机制可能影响mTOR mRNA的稳定性，从而影响其活性。此外，这一机制可以影响更广泛的mRNA，并作为一种通用机制，以SAM68依赖的方式调节mRNA的剪接和稳定性。这将通过以下方式实现： 目的1：确定SAM68募集关键剪接体成分和调控剪接机制的机制。 目的2：明确SAM68如何调节mTOR mRNA的稳定和降解。 在这个拟议的研究计划中，我们将定义一个新发现的SAM68--影响选择性剪接和mRNA稳定性的调控机制。虽然我们的大部分研究将使用我们的mTOR模型启动，但我们也将确定这种SAM68调节的机制是否会影响其他可能作为涉及细胞完整性的新的翻译保障机制的mRNA。

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.