Functional characterization of the TTF complex and its role in neurodevelopmental disorders
Functional characterization of the TTF complex and its role in neurodevelopmental disorders
复制标题
DOI：
10.25972/opus-15778
复制
发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Cornelia Brosi
中科院分区：
文献类型：
--
作者：
Cornelia Brosi
真核基因的表达需要广泛的调节，以使细胞的稳态，并允许由于外部刺激的动态响应。尽管许多调控机制涉及作为基因表达的第一步的转录，但转录后mRNA代谢也发生了强烈的调控。因此，mRNP的特定组成起着核心作用，因为与mRNA相关的组分形成决定mRNA命运的特定“mRNP密码”。参与这种调节和mRNA代谢的许多蛋白质在疾病中受到影响，特别是神经系统疾病通常由异常mRNP编码引起，其导致mRNP的调节和表达的变化。本工作的重点是基于其亚基TDRD 3、TOP 3 β和FMRP的三聚体蛋白复合物，称为TTF复合物。生物化学研究表明，TTF复合物的三种组分是核-胞质穿梭蛋白，其在稳态时定位于细胞质中，与mRNP相关联，并且推测与翻译有关。在细胞应激条件下，TTF组分集中在应激颗粒中。因此，TTF复合物是mRNP密码的一部分，但其靶RNA和功能仍然完全未知。由于功能性FMRP的丧失会导致脆性X综合征，而TOP3β与精神分裂症和智力残疾有关，因此TTF复合体在分子水平上将这些表型相关的神经精神疾病相互联系起来。因此，这项工作的目的是生物化学特性的TTF复合物，并确定其在mRNA代谢的功能。在这项工作中，提供了TDRD 3作为TTF复合物的中心单元并直接结合FMRP以及TOP3β的证据。因此，TDRD 3和TOP 3 β的相互作用是非常稳定的，而FMRP是动态组分。有趣的是，TTF复合物不直接与mRNA结合，而是通过外显子连接复合物（EJC）募集到mRNP。这种相互作用由TDRD 3的特异性结合基序EBM介导。通过生物化学和生物学研究，可以鉴定TTF复合物的相互作用组并确定其在mRNA代谢中的作用。数据显示，TTF复合物主要与“早期”mRNP相关，可能参与了翻译的先锋轮。此外，TOP3β被发现直接与核糖体结合，从而在EJC和翻译机器之间建立了联系。TTF组分的减少导致了培养细胞中蛋白质组的选择性变化，由此个体蛋白质亚群似乎受到调节，而不是全局蛋白质表达。对TOP3β的酶学分析表明，TOP3β是一种IA型拓扑异构酶，它不仅能催化DNA，而且能催化RNA。关于早期mRNP和这项工作中揭示的翻译之间的联系，这方面特别有趣。在这项工作中获得的数据表明，TTF复合物在调节代谢的早期mRNP子集可能在翻译的先锋轮的过程中发挥作用。到目前为止，RNA拓扑异构酶和mRNA代谢之间的联系是独特的，因此提供了一个全新的视角，在转录后基因表达及其调控的步骤。
The eukaryotic gene expression requires extensive regulations to enable the homeostasis of the cell and to allow dynamic responses due to external stimuli. Although many regulatory mechanisms involve the transcription as the first step of the gene expression, intensive regulation occurs also in the post-transcriptional mRNA metabolism. Thereby, the particular composition of the mRNPs plays a central role as the components associated with the mRNA form a specific “mRNP code” which determines the fate of the mRNA. Many proteins which are involved in this regulation and the mRNA metabolism are affected in diseases and especially neurological disorders often result from an aberrant mRNP code which leads to changes in the regulation and expression of mRNPs. The focus of this work was on a trimeric protein complex which is termed TTF complex based on its subunits TDRD3, TOP3β and FMRP. Biochemical investigations revealed that the three components of the TTF complex are nucleo-cytosolic shuttle proteins which localize in the cytoplasm at the steady-state, associate with mRNPs and are presumably connected to the translation. Upon cellular stress conditions, the TTF components concentrate in stress granules. Thus, the TTF complex is part of the mRNP code, however its target RNAs and function are still completely unknown. Since the loss of functional FMRP results in the fragile X syndrome and TOP3β is associated with schizophrenia and intellectual disability, the TTF complex connects these phenotypically related neuro-psychiatric disorders with each other on a molecular level. Therefore, the aim of this work was to biochemically characterize the TTF complex and to define its function in the mRNA metabolism. In this work, evidence was provided that TDRD3 acts as the central unit of the TTF complex and directly binds to FMRP as well as to TOP3β. Thereby, the interaction of TDRD3 and TOP3β is very stable, whereas FMRP is a dynamic component. Interestingly, the TTF complex is not bound directly to mRNA, but is recruited via the exon junction complex (EJC) to mRNPs. This interaction is mediated by a specific binding motif of TDRD3, the EBM. Upon biochemical and biological investigations, it was possible to identify the interactome of the TTF complex and to define the role in the mRNA metabolism. The data revealed that the TTF complex is mainly associated with “early” mRNPs and is probably involved in the pioneer round of translation. Furthermore, TOP3β was found to bind directly to the ribosome and thus, establishes a connection between the EJC and the translation machinery. A reduction of the TTF components resulted in selective changes in the proteome in cultured cells, whereby individual protein subsets seem to be regulated rather than the global protein expression. Moreover, the enzymatic analysis of TOP3β indicated that TOP3β is a type IA topoisomerase which can catalytically attack not only DNA but also RNA. This aspect is particularly interesting with regard to the connection between early mRNPs and the translation which has been revealed in this work. The data obtained in this work suggest that the TTF complex plays a role in regulating the metabolism of an early mRNP subset possibly in the course of the pioneer round of translation. Until now, the link between an RNA topoisomerase and the mRNA metabolism is thereby unique and thus provides a completely new perspective on the steps in the post-transcriptional gene expression and its regulation.