RBM4藉由調控丙酮酸激酶之選擇性剪接 促進間質性幹細胞之神經分化

RBM4 regulates neuronal differentiation of mesenchymal stem cells by modulating alternative splicing of Pyruvate kinase M

蘇俊豪(Chun-Hao Su)

指導教授 ： 譚婉玉

國立臺灣大學/醫學院/分子醫學研究所/博士(2017年)

https://doi.org/10.6342/NTU201701122

摘要

mRNA 的選擇性剪接對大腦的發育以及神經功能有重要的影響。RBM4可透過調控選擇性剪接促進神經前驅細胞的分化及培養中神經細胞突觸的生長。本論文中，我進一步的探討RBM4在神經分化過程中扮演的角色。在神經分化過程中，能量產生路徑由醣解作用改變為氧化磷酸化作用。我發現在腦部發育過程中， 醣解代謝酵素丙酮酸激酶(PKM)的選擇性剪接會使酵素由M2型變為M1型。同時，我觀察到在小鼠胚胎腦中，單一Rbm4 基因的剔除會影響此酵素異構物的轉變。在間質性幹細胞(Mesenchymal stem cells; MSCs)的神經分化過程中，亦會發生此一選擇性剪接作用。利用PKM minigene，我證實RBM4能直接調控此一選擇性剪接作用。在MSC中過量表達RBM4或是PKM1可以誘導神經相關基因的表現，同時亦能增加細胞的粒線體呼吸作用並促進神經性分化。因此，在神經分化過程中，RBM4 對於PKM異構型的轉變以及其導致的能量代謝作用之改變扮演著重要的角色。此外，RBM4所調控之PKM異構物轉變，也可能進一步影響細胞增生以及細胞內醣解作用相關酵素的表現。另外，此研究中我們亦發現在低氧促進MSC神經性分化過程中，RBM4被誘導而增加其表現，並且進一步調控PKM異構物的轉變以及神經相關基因的表達。此結果顯示RBM4具有應用於神經性退化疾病治療的潛力。最後，我發現了在不同型態的細胞中，RBM4藉由不同的機制影響另一個選擇性剪接調控分子PTB的表現。我證實了在MSC中，RBM4可以拮抗PTB的作用並且影響其表現而產生一個具較弱抑制性的PTB異構物。因此，RBM4透過改變不同選擇性剪接作用而在神經分化過程中扮演著重要的角色。本論文再證明了選擇性剪接在神經性分化或者是大腦發育過程中之重要性。

關鍵字

選擇性剪接 ； 低氧 ； 間質性幹細胞 ； 神經性分化 ； 丙酮酸激酶

並列摘要

Brain development and neurologic functions involve numerous alternative splicing (AS) events. RBM4 promotes differentiation of neuronal progenitor cells and neurite outgrowth of cultured neurons via its role in splicing regulation. In this study, we further explored the role of RBM4 in neuronal differentiation. During neuronal differentiation, energy production shifts from glycolysis to oxidative phosphorylation. We found that the splice isoform change of the metabolic enzyme pyruvate kinase M (PKM) from PKM2 to PKM1 occurs during brain development and is impaired in RBM4-deficient brains. The PKM isoform change could be recapitulated in human mesenchymal stem cells (MSCs) during neuronal induction. Using a PKM minigene, we demonstrated that RBM4 plays a direct role in regulating alternative splicing of PKM. Overexpression of RBM4 or PKM1 induced the expression of neuronal genes, increased mitochondrial respiration capacity in MSCs, and, accordingly, promoted neuronal differentiation. Hence, RBM4 plays an important role in the PKM isoform switch and change in mitochondrial energy production during neuronal differentiation. In addition, RBM4-mediated isoform switch of PKM may affect cell proliferation and the expression of glycolytic enzymes. Moreover, we demonstrated that RBM4 is induced and is involved in the PKM splicing switch and neuronal gene expression during hypoxia-induced neuronal differentiation. These results reveal the potential of RBM4 in therapeutic application of neurodegenerative diseases. Finally, RBM4 regulates the expression of another critical splicing regulator PTB, which involves distinct mechanism among different cell types. We demonstrated that RBM4 antagonized the function of PTB and induced the expression of a PTB isoform with attenuated splicing activity in MSCs. Thus, RBM4 modulates neuronal differentiation essentially via its role in alternative splicing.

並列關鍵字

Alternative splicing ； hypoxia ； mesenchymal stem cells ； neuronal differentiation ； pyruvate kinase M

參考文獻

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