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

拓扑异构酶被认为是主要作用于所有王国细胞的DNA底物上的酶，如果不是唯一的话。因此，令人惊讶的是，包括我们在内的两个实验室提供的证据表明，真核拓扑异构酶top3？是一种细胞质RNA结合蛋白，在体外对RNA底物具有酶活性。我们的生化研究进一步表明，在脆性X智力低下综合征(FXS)中，top3ü与支架蛋白TDRD3和FMRP一起形成，这是一种体内未知的三聚体复合体。我们将这个单元命名为top3？-TDRD3-FMRP(TTF)复合体，它通过外显子连接复合体(EJC)以不依赖于序列的方式被招募到mRNPs。含有TTF的mRNPs的生化组成及其与多聚体的关联进一步表明，它们参与了第一轮翻译。然而，TTF复合体的体内RNA靶点及其对RNA的调节作用方式尚不清楚。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的病因可能相交于与top3？和FMRP相关的RNA分子的转录后调控。本项目旨在通过a)确定TTF复合体在体内的RNA靶标和b)确定其在RNA代谢中的催化和/或调节功能来阐明TTF在RNA代谢中的作用。这些研究还将有助于c)阐明神经精神障碍的潜在的基于RNA的病因学。

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.