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Functional Characterization of the TTF Complex and Its Role in Neurodevelopmental Disorders

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Functional Characterization of the TTF Complex and Its Role in Neurodevelopmental Disorders Functional characterization of the TTF complex and its role in neurodevelopmental disorders Doctoral Dissertation (Thesis) to obtain the degree of “Doktor der Naturwissenschaften” (Dr. rer. nat.) awarded by the Julius-Maximilians-Universität Würzburg submitted by Cornelia Brosi from Würzburg, Germany Würzburg 2017 Submitted to the Faculty of Chemistry and Pharmacy on December 21, 2017 Evaluators of the written Dissertation (Thesis) Supervisor – 1. Evaluator: Prof. Dr. Utz Fischer 2. Evaluator: Prof. Dr. Alexander Buchberger Examiners of the Public Defense 1. Examiner: Prof. Dr. Utz Fischer 2. Examiner: Prof. Dr. Alexander Buchberger 3. Examiner: PD Dr. Sibylle Jablonka Date of the Public Defense Doctoral Certificate awarded on Table of contents 1. Summary.......................................................................................................................................... 1 2. Zusammenfassung ........................................................................................................................... 3 3. Introduction ..................................................................................................................................... 5 3.1. mRNA processing - an important step in the eukaryotic gene expression .................................. 5 3.2. The mRNP code regulates the post-transcriptional gene expression .......................................... 7 3.3. mRNA-binding proteins .............................................................................................................. 10 3.4. The mRNA metabolism and neuropsychiatric diseases ............................................................. 11 4. Results ........................................................................................................................................... 20 4.1. TDRD3 forms a complex with FMRP and TOP3β and binds directly to the EJC ......................... 20 4.2. Identifying the TTF complex interactome I: biochemical purifications ...................................... 22 4.3. Identifying the TTF complex interactome II: mass spectrometric analysis ................................ 24 4.4. TDRD3 associates with mRNPs and polysomes independently of its interaction with TOP3β and FMRP ................................................................................................................................................. 30 4.5. TDRD3 and TOP3β are tightly connected and associate with the pioneer round of translation 34 4.6. TOP3β binds directly to the 80S ribosome via its RGG box ........................................................ 37 4.7. TOP3β can recruit TDRD3 to the 80S ribosome ......................................................................... 39 4.8. TOP3β binds to rRNA of the 40S and the 60S ribosomal subunits ............................................. 41 4.9. TOP3β is a potential RNA topoisomerase but associates with polysomes independently of its catalytical activity and ribosome binding .......................................................................................... 43 4.10. TDRD3, TOP3β and FMRP regulate the expression of a subset of proteins ............................. 46 5. Discussion ...................................................................................................................................... 50 5.1. TDRD3 is the central hub in the formation of the TTF complex and its binding to the EJC ....... 51 5.2. Contribution of TOP3β and FMRP to the cellular interactions of the TTF complex ................... 54 5.3. The TTF interactome reflects early mRNPs and the translation machinery and links the TTF complex to the pioneer round of translation .................................................................................... 55 5.4. TDRD3 and TOP3β form a stable-subunit within the TTF complex ............................................ 58 5.5. TOP3β directly binds to the 80S ribosome and connects also TDRD3 with the ribosome ........ 59 5.6. TOP3β is an RNA topoisomerase bound to ribosomal RNA ....................................................... 60 5.7. The TTF components impact the expression of a subset of proteins ........................................ 63 5.8. Potential functions of the TTF complex in the mRNA metabolism of early mRNPs................... 64 6. Material and methods ................................................................................................................... 70 6.1. Material ...................................................................................................................................... 70 6.1.1. Protein and nucleotide ladders ............................................................................................... 70 6.1.2. Standard buffers ...................................................................................................................... 70 6.1.3. Cell culture ............................................................................................................................... 72 6.1.3.1. organisms and strains ........................................................................................................... 72 6.1.3.2. Cell culture media................................................................................................................. 74 6.1.4. Antibodies ............................................................................................................................... 76 6.1.5. Plasmid vectors ....................................................................................................................... 77 6.1.6. DNA and RNA oligonucleotides ............................................................................................... 79 6.2. Methods ..................................................................................................................................... 80 6.2.1. Molecular biological methods ................................................................................................. 80 6.2.2. Eukaryotic cell culture ............................................................................................................. 83 6.2.3. Biochemical methods .............................................................................................................. 86 6.2.4. Immunobiochemical methods ................................................................................................ 94 7. Supplementary information ........................................................................................................ 96 8. Bibliography ................................................................................................................................. 101 9. Table of figures ............................................................................................................................ 110 11. Acknowledgements ................................................................................................................... 112 12. Declaration ................................................................................................................................ 113 1. Summary 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
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