【作者】 刘敏；

【导师】 周强； 陈瑞川；

【作者基本信息】 厦门大学 ， 细胞生物学， 2008， 博士

【摘要】 在真核细胞中,编码蛋白的基因的转录均由RNA聚合酶Ⅱ(RNA PolⅡ)来完成,其转录过程大致由起始前复合物组装、起始、启动子区清扫、延伸和终止等紧密衔接的步骤组成。近年来研究发现原先最不受重视的转录延伸步骤其实是细胞调控转录最重要、也最复杂的调控平台。其中正性转录延伸因子b复合体(positive transcription elongation factor b,P-TEFb复合体)被证明是协调转录延伸步骤的最重要的因子之一。P-TEFb复合体是由CDK9及CyclinT1组成的异二聚体激酶,在转录延伸过程中可磷酸化RNA polⅡ大亚基CTD上第2位丝氨酸,并克服负性延伸因子对转录延伸的抑制作用,从而保证全长mRNA的转录。作为基本转录因子,P-TEFb复合体不仅是细胞内绝大多数基因转录所必需的因子,其活性的异常调控与心肌肥大和癌症等疾病也有直接或间接的关联。更重要的是,P-TEFb复合体也是艾滋病病毒(HIV-1)转录复制过程中所必需的宿主细胞因子之一。本实验室的前期研究揭示了P-TEFb复合体活性调控的分子模式:即核蛋白HEXIM1通过核小RNA 7SK snRNA与P-TEFb复合体结合并形成无活性的7SK snRNP复合物,任何导致该复合物解离的因素均可活化P-TEFb复合体的转录活性。因此,了解细胞内调控P-TEFb复合体活化的信号途径和分子机制对于了解上述重大疾病的病因具有重要的科学意义。但迄今为止,对调控P-TEFb复合体活化的信号途径和分子机制仍知之甚少。本文针对调控P-TEFb复合体活化的信号途径及其分子机制展开研究,发现P-TEFb复合体的活化需通过两条不同的信号途径之间的协同作用来完成。首先,为探讨P-TEFb复合体活化的信号途径,我们对能够诱导P-TEFb复合体活化的胞外刺激因子进行了筛选,发现DNA损伤诱导因子、缺氧条件、酸性或细胞分化诱导剂HMBA等因子均能激发P-TEFb复合体的活化。提示P-TEFb复合体似乎作为多种胞内重要的生理信号途径的整合节点,协调并影响生理或病理转变中的特异性转录。采用UV和HMBA这两种经典的P-TEFb复合体活化因子处理HeLa细胞,发现均能引发HeLa细胞的钙离子内流,其后采用多种信号途径特异性抑制剂和分子生物学手段抑制或增强钙信号途径均可相应地拮抗或激发P-TEFb复合体的活化,从而证实钙离子/PP2B(calcineurin)信号通路是应答胞外刺激、调控P-TEFb复合体活化所必需的信号途径。但之后又发现该途径单独并不能直接活化P-TEFb复合体,提示需要第二条信号通路的协同作用。随后鉴定发现蛋白磷酸酶PP1α信号途径也是必需的途径之一,而且和钙离子/PP2B途径相类似,PP1α途径单独同样也不能起作用。采用共转染和共处理等分子生物学及生物化学方法,我们发现PP2B和PP1α在体内和体外均能直接并且有效地协同活化P-TEFb复合体,同时发现PP2B的去磷酸化作用必需先于PP1α的去磷酸化作用,表明两条不同的信号途径所介导的两步去磷酸化是活化P-TEFb复合体的关键机制。采用特制的抗磷酸化抗体,证实CDK9 T-loop上磷酸化的Thr186(pT186)是由PP1α去磷酸化的,并且发现该位点的去磷酸化是调控P-TEFb复合体活化的关键位点。我们的研究结果表明细胞外刺激因子可通过细胞内钙离子/PP2B及PP1α两条主要信号途径传导,并通过协同作用而激活P-TEFb复合体的活性,其中PP2B的去磷酸化作用可能是将CDK9 T-loop上的pT186暴露给PP1α并由PP1α对该位点进行去磷酸化,最终导致7SK snRNP复合物的解离而解除对P-TEFb复合体的抑制,从而激活P-TEFb复合体的转录活性。更多还原

【Abstract】 In eukaryotes, the transcription of protein-coding genes is performed by RNA polymeraseⅡin a process consisting of several tightly connected stages designated as pre-initiation, initiation, promoter clearance, elongation and termination. The accumulating evidence has substantially demonstrated that the elongation stage, an ignored stage in the past, is, in fact, the most important and complex platform for the regulation of transcription. Among the numerous of transcription related factors, the positive transcription elongation factor b (P-TEFb) has emerged as the one of the most important factor functioning in control of elongation stage. Consisting of CDK.9 and its regulator Cyclin T1, P-TEFb functions as an RNA polymerase (Pol)Ⅱspecific kinase responsible for the phosphorylation of second serine residue (Ser2) of C-terminal domain of PolⅡ. This phosphorylation is key important for stimulation of PolⅡelongation activity to ensure the synthesis of full-length pre-mRNA. As a general transcription factor, P-TEFb not only is required for the transcription of most mRNA-encoding genes, but also is indispensable for the development of embryo, Moreover, P-TEFb also is a cellular co-factor essential for AIDS virus (HIV-1) replication. The abnormal regulation of P-TEFb activity has been demonstrated as a pathogenesis factor of cardiac hypertrophy and tumor. Our previous reports have established the P-TEFb activity regulation model: the nuclear protein HEXIM1 binds to and inhibits P-TEFb activity with 7SK snRNA as scaffold, and the release of P-TEFb from this inactive 7SK snRNP complex means the activation of P-TEFb. However, how the cell releases the P-TEFb from the association of HEXIM1/7SK snRNA to activate its activity is still remaining largely unknown.Reported here, we demonstrate that the activation of cellular P-TEFb activity is accomplished by the cooperative actions of two major signaling pathways. To explore the signal pathway(s) responsible for mediating the disruption of inactive 7SK snRNP complex, we firstly treated cells with different kinds of agents to screen the efficient treating model. Besides UV irradiation and HMBA short term treatment, we found that the other stimulation clues such as DNA damage-inducing agents, hypoxia-inducing agents, etc, could also induce the disruption of the inactive 7SK snRNP complex, suggesting that P-TEFb may serve as an integrating point of different signal pathways.By irradiation with UV and treatment with HMBA, the two classic agents used for disruption the inactive 7SK snRNP complex, we found the strong calcium influx caused by UV and HMBA in HeLa cells. With the pharmacological and molecular approaches, we demonstrated that calcium/PP2B signaling pathway is an essential but not sufficient pathway for mediating the UV- and HMBA-induced disruption of the inactive 7SK snRNP complex. Subsequently, we found that PP1αsignal pathway is also a necessary, however, insufficient pathway for mediating UV- and HMBA-induced disruption of the inactive complex. By using the co-transfection and co-treatment methods, we finally demonstrated that PP2B and PP1αmust function cooperatively and only when PP2B functions prior to PP1αto disrupt the inactive 7SK snRNP complex. With an antibody specifically against phosphor-Thr186 (pT186) of CDK9, we demonstrated that PP1αcould dephosphorylate the pT186 of CDK9, however, only accomplished when PP1αworked together with PP2B or more directly, when the 7SK snRNP was disrupted by RNase treating. Furthermore, this dephosphorylation was proven, both in vitro and in vivo, as key important for releasing P-TEFb from inactive 7SK snRNP complex. Token together, our data suggest a two-stepwise mechanism for the activation of P-TEFb, in which the function of PP2B may only serve as an accessorial factor causing the conformation change of inactive 7SK snRNP complex via the dephosphorylation on unknown phosphor-residue and make the pT186 of CDK9 accessible for PP1αwhich finally dephosphorylates pT186 of CDK9 to release the P-TEFb from inhibitory complex.更多还原