RNA methylation and mesenchymal stem cell differentiation

项目摘要 间充质干细胞(MSCs)可以发育为成骨细胞、脂肪细胞和软骨细胞，为 再生医学材料。特别是，骨髓间充质干细胞的骨相关应用是最有前途的应用之一。 骨髓间充质干细胞的临床应用。PI最近发现FK506结合蛋白(FkBP)的成员Fkbp4 PPIase家族，促进MSC向成骨细胞分化。他们还发现， Fkbp4与METTL3复合体相互作用，从而诱导一种名为N6-甲基腺苷的新的RNA修饰 (M6A)。虽然已知m6A参与了间充质干细胞的分化，但确切的作用和机制在很大程度上仍然存在 未知。通过全基因组的方法，Pi在MSCs中发现了数千个被m6A修饰的mRNA， 成骨细胞和脂肪细胞。这些mRNAs还包括分化的关键转录因子基因。 作为几个组蛋白修饰酶基因。此外，他们还发现，Fkbp4激活了METTL3复合体 一种依赖于PPIase域的方式。根据这些发现，PI假设Fkbp4激活了 在成骨细胞分化过程中，METTL3通过其一个亚基的异构化形成复合体。他们还假设 M6A修饰通过调节RNA代谢促进成骨细胞分化 提高了基因的蛋白质水平。PI将通过以下三个目标来检验这些假设。在目标1中， PI将在单个核苷酸上定位m6A在MSCs、成骨细胞和脂肪细胞转录组中的分布 水平。随后，它们将以序列特异性的方式抑制甲基化，以了解原因 M6A与RNA代谢的关系。目的2将研究Fkbp4基因敲除的骨表型 并研究成骨细胞基因m6A如何影响它们的分化。AIM 3将研究M6A修饰 Fkbp4作为Fkbp4和METTL3之间的反馈。此外，这一目标将调查Fkbp4如何 糖皮质激素受体抑制脂肪细胞分化过程中的表达。总的来说，这些研究将 展示Fkbp4对METTL3的一种新的调控机制以及m6A修饰如何控制MSC 差异化。这些发现有望促进以MSC为基础的再生医学。

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.