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

SAM68 通过 mTOR mRNA 剪接和稳定诱导细胞代谢调节

• 批准号: RGPIN-2019-06494
• 负责人: Huot, MarcÉtienne
• 金额: $ 2.33万
• 依托单位: Université Laval
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=745056
• 关键词: SAM68; induced; regulation; cellular; metabolism

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.

细胞代谢的调节对于正常细胞生长、体内平衡、完整性和存活至关重要。它涉及由雷帕霉素（mTOR）信号通路的机制靶点协调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，并且已知调节细胞生长、细胞增殖、细胞运动性、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研究计划的初始阶段，我们将SAM 68确定为mTOR剪接的关键调节因子。我们发现Sam 68缺失引起内含子5保留增加，产生称为mTORi 5的较小mRNA。虽然确切的分子机制仍然未知，但我们最近的研究表明，SAM 68可以与剪接体的核心组分U1 snRNP相互作用，这有助于5'剪接位点的识别。这表明SAM 68和剪接体组分之间的相互作用可能是mTOR正常剪接和正确剪接位点识别所必需的。此外，虽然可以检测到mTORi 5 mRNA，但没有检测到对应于该特定转录物的蛋白产物。尽管如此，mTORi 5 mRNA变体的表达增加与mTOR全长蛋白表达的急剧降低相关，表明Sam 68具有通过mTOR前体mRNA的特异性可变剪接检查点调节mTOR表达的能力。我们的假设是，这种SAM 68调节的剪接机制可能影响mTOR mRNA的稳定性，从而影响其活性。此外，这种机制可以影响更广泛的mRNA，并作为一种通用机制，以SAM 68依赖的方式调节mRNA剪接和稳定性。这将通过以下方式实现：目的1：确定SAM 68可以募集关键剪接体组分并调节剪接机制的机制。目的2：确定SAM 68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SAM 68调节机制，影响选择性剪接和mRNA的稳定性。虽然我们的大部分研究将使用我们的mTOR模型启动，但我们还将确定这种SAM 68调节机制是否会影响其他mRNA，这些mRNA可能作为参与细胞完整性的新型翻译保护机制。