Functional analysis of the TTF complex and its role in neurodevelopmental diseases

拓扑异构酶是被认为在所有界的细胞中主要（如果不是排他的话）作用于DNA底物的酶。因此，当包括我们在内的两个实验室提供证据证明真核拓扑异构酶TOP3 β是一种细胞溶质RNA结合蛋白，在体外对RNA底物表现出酶活性时，这是一个惊喜。我们的生物化学研究进一步表明，TOP3 β与TDRD 3（一种支架蛋白）和FMRP一起形成，影响脆性X智力低下综合征（FXS），这是一种未知的体内三聚体复合物。我们将该单元命名为T0 P3 β-TDRD 3-FMRP（TTF）复合物，其以不依赖于序列的方式经由外显子连接复合物（EJC）被募集到mRNP。含有TTF的mRNP的生化组成及其与多聚核糖体的结合进一步表明它们参与了翻译的先锋轮。然而，TTF复合物的体内RNA靶点及其对RNA的调节作用模式都是未知的。有趣的是，TOP3基因的缺失与精神分裂症和智力残疾有关。因此，精神分裂症和FXS的病因学可能在与TOP 3 β和FMRP相关的RNA分子的转录后调节上交叉。本项目旨在阐明TTF在RNA代谢中的作用，通过a）在体内鉴定TTF复合物的RNA靶点和B）确定其在RNA代谢中的催化和/或调节功能。这些研究还将有助于c）阐明神经精神疾病的潜在RNA病因。

Topoisomerases are enzymes believed to act predominantly, if not exclusively, on DNA substrates in cells of all kingdoms. It was therefore a surprise when two labs, including ours, provided evidence that eukaryotic topoisomerase TOP3ß is a cytosolic RNA binding protein that exhibits enzymatic activity on RNA substrates in vitro. Our biochemical studies have further shown that TOP3ß forms together with TDRD3, a scaffolding protein, and FMRP, affected in Fragile X mental retardation syndrome (FXS), an as yet unknown trimeric complex in vivo. This unit, which we have named TOP3ß-TDRD3-FMRP (TTF) complex, is recruited to mRNPs via the exon junction complex (EJC) in a sequence independent manner. The biochemical composition of TTF-containing mRNPs and their association with polysomes further suggests that they are engaged in the pioneer round of translation. However, neither the in vivo RNA targets of the TTF complex nor its regulatory mode of action on RNA are known. Interestingly, deletion of the TOP3ß-gene is associated with schizophrenia and intellectual disability. It is therefore possible that the etiologies of schizophrenia and FXS intersect at the post-transcriptional regulation of RNA molecules associated with TOP3ß and FMRP. This project aims to elucidate the role of TTF in RNA metabolism by a) identifying the RNA targets of the TTF complex in vivo and b) determining its catalytic and/or regulatory function in RNA metabolism. These studies will also help to c) shed light on a potential RNA-based etiology of neuropsychiatric disorders.