Functional characterization of the TTF complex and its role in neurodevelopmental disorders

Abstract

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.