RNA methylation and mesenchymal stem cell differentiation

项目摘要 间充质干细胞（MSC）可以发育成骨细胞、脂肪细胞和软骨细胞， 再生医学的材料。尤其是骨髓间充质干细胞在骨相关领域的应用是最有前景的 MSCs的临床应用PI最近发现，Fkbp 4，FK 506结合蛋白（Fkbp）的成员， 肽基脯氨酰异构酶（PPI酶）家族，促进MSC分化成骨细胞。他们还发现 Fkbp 4与Mettl 3复合物相互作用，诱导称为N6-甲基腺苷的新RNA修饰 （m6A）。虽然已知m6 A参与MSC分化，但确切的作用和机制在很大程度上仍然存在 未知通过全基因组方法，PI在MSC中发现了数千种m6 A修饰的mRNA， 成骨细胞和脂肪细胞。这些mRNA还包括分化的关键转录因子基因 作为几种组蛋白修饰酶基因。此外，他们发现Fkbp 4激活Mettl 3复合物， PPIase结构域依赖的方式。基于这些发现，PI假设Fkbp 4激活 通过在成骨细胞分化过程中其一个亚基的异构化而形成的Mettl 3复合物。他们还假设 m6 A修饰通过调节RNA代谢促进成骨细胞分化， 增加基因的蛋白质水平。PI将通过以下三个目标检验这些假设。在目标1中， PI将绘制m6 A在MSC、成骨细胞和脂肪细胞转录组中的单核苷酸分布图， 水平随后，他们将以序列特异性方式抑制甲基化，以了解因果关系。 m6 A与RNA代谢的关系目的2将研究Fkbp 4基因敲除后的骨表型 小鼠，并研究成骨细胞基因m6 A如何影响其分化。目标3将研究m6 A修饰 Fkbp 4 mRNA的表达作为Fkbp 4和Mettl 3之间的反馈。此外，本研究还将探讨Fkbp 4 糖皮质激素受体在脂肪细胞分化过程中抑制表达。这些研究将 证明了Fkbp 4对Mettl 3的新的调节机制以及m6 A修饰如何控制MSC 分化这些发现有望促进基于MSC的再生医学。

Project Summary Mesenchymal stem cells (MSCs) can develop into osteoblasts, adipocytes, and chondrocytes, providing materials for regenerative medicine. In particular, bone-related applications of MSCs is one of the most promising clinical applications of MSCs. The PI recently found that Fkbp4, a member of the FK506-binding protein (Fkbp) family of peptidyl prolyl isomerase (PPIase), promotes MSC differentiation into osteoblasts. They also found that Fkbp4 interacts with the Mettl3 complex, which induces the novel RNA modification called N6-methyladenosine (m6A). Although m6A is known to be involved in MSC differentiation, exact roles and mechanisms remain largely unknown. Through a genome-wide approach, PI found thousands of mRNAs modified by m6A in MSCs, osteoblasts, and adipocytes. The mRNAs included critical transcription factor genes for the differentiation as well as several histone modifying enzyme genes. In addition, they found that Fkbp4 activates the Mettl3 complex in a PPIase domain-dependent manner. Based on these findings, the PI hypothesized that Fkbp4 activates the Mettl3 complex by isomerization of one of its subunits during osteoblast differentiation. They also hypothesized that m6A modifications promote osteoblast differentiation by modulating RNA metabolism with a result of increased protein levels of the genes. The PI will test these hypotheses with the following three aims. In Aim 1, the PI will map m6A distributions in the transcriptome of MSCs, osteoblasts, and adipocytes at a single nucleotide level. Subsequently, they will inhibit the methylation in a sequence-specific manner to understand causal relationships between m6A and RNA metabolism. Aim 2 will investigate bone phenotypes of Fkbp4 knockout mice and also study how m6A of osteoblast genes affect their differentiation. Aim 3 will study m6A modification of Fkbp4 mRNA as a feedback between Fkbp4 and Mettl3. In addition, this aim will investigate how Fkbp4 expression is inhibited during adipocyte differentiation by glucocorticoid receptor. Collectively, these studies will demonstrate a novel regulatory mechanism of Mettl3 by Fkbp4 and how m6A modifications controls MSC differentiation. These findings are expected to promote MSC-based regenerative medicine.