@phdthesis{Wanzek2016,
  author    = {Wanzek, Katharina},
  title     = {The investigation of the function of repair proteins at G-quadruplex structures in \(Saccharomyces\) \(cerevisiae\) revealed that Mms1 promotes genome stability},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142547},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {G-quadruplex structures are highly stable alternative DNA structures that can, when not properly regulated, impede replication fork progression and cause genome instability (Castillo Bosch et al, 2014; Crabbe et al, 2004; Koole et al, 2014; Kruisselbrink et al, 2008; London et al, 2008; Lopes et al, 2011; Paeschke et al, 2013; Paeschke et al, 2011; Piazza et al, 2015; Piazza et al, 2010; Piazza et al, 2012; Ribeyre et al, 2009; Sabouri et al, 2014; Sarkies et al, 2012; Sarkies et al, 2010; Schiavone et al, 2014; Wu \& Spies, 2016; Zimmer et al, 2016). The aim of this thesis was to identify novel G-quadruplex interacting proteins in Saccharomyces cerevisiae and to unravel their regulatory function at these structures to maintain genome integrity. Mms1 and Rtt101 were identified as G-quadruplex binding proteins in vitro via a pull-down experiment with subsequent mass spectrometry analysis. Rtt101, Mms1 and Mms22, which are all components of an ubiquitin ligase (Rtt101Mms1/Mms22), are important for the progression of the replication fork following fork stalling (Luke et al, 2006; Vaisica et al, 2011; Zaidi et al, 2008). The in vivo binding of endogenously tagged Mms1 to its target regions was analyzed genome-wide using chromatin-immunoprecipitation followed by deep-sequencing. Interestingly, Mms1 bound independently of Mms22 and Rtt101 to G-rich regions that have the potential to form G-quadruplex structures. In vitro, formation of G-quadruplex structures could be shown for the G-rich regions Mms1 bound to. This binding was observed throughout the cell cycle. Furthermore, the deletion of MMS1 caused replication fork stalling as evidenced by increased association of DNA Polymerase 2 at Mms1 dependent sites. A gross chromosomal rearrangement assay revealed that deletion of MMS1 results in a significantly increased genome instability at G-quadruplex motifs compared to G-rich or non-G-rich regions. Additionally, binding of the helicase Pif1, which unwinds G4 structures in vitro (Paeschke et al, 2013; Ribeyre et al, 2009; Sanders, 2010; Wallgren et al, 2016), to Mms1 binding sites was reduced in mms1 cells. The data presented in this thesis, together with published data, suggests a novel mechanistic model in which Mms1 binds to G-quadruplex structures and enables Pif1 association. This allows for replication fork progression and genome integrity.},
  subject      = {Quadruplex-DNS},
  language  = {en}
}
@article{WaldholmWangBrodinetal.2011,
  author    = {Waldholm, Johan and Wang, Zhi and Brodin, David and Tyagi, Anu and Yu, Simei and Theopold, Ulrich and {\"O}stlund Farrants, Ann Kristin and Visa, Neus},
  title     = {SWI/SNF regulates the alternative processing of a specific subset of pre-mRNAs in \(Drosophila\) \(melanogaster\)},
  series = {BMC Molecular Biology},
  volume    = {12},
  journal   = {BMC Molecular Biology},
  number    = {46},
  doi       = {10.1186/1471-2199-12-46},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142613},
  pages     = {1-12},
  year      = {2011},
  abstract  = {Background: The SWI/SNF chromatin remodeling factors have the ability to remodel nucleosomes and play essential roles in key developmental processes. SWI/SNF complexes contain one subunit with ATPase activity, which in Drosophila melanogaster is called Brahma (Brm). The regulatory activities of SWI/SNF have been attributed to its influence on chromatin structure and transcription regulation, but recent observations have revealed that the levels of Brm affect the relative abundances of transcripts that are formed by alternative splicing and/or polyadenylation of the same pre-mRNA. Results: We have investigated whether the function of Brm in pre-mRNA processing in Drosophila melanogaster is mediated by Brm alone or by the SWI/SNF complex. We have analyzed the effects of depleting individual SWI/SNF subunits on pre-mRNA processing throughout the genome, and we have identified a subset of transcripts that are affected by depletion of the SWI/SNF core subunits Brm, Snr1 or Mor. The fact that depletion of different subunits targets a subset of common transcripts suggests that the SWI/SNF complex is responsible for the effects observed on pre-mRNA processing when knocking down Brm. We have also depleted Brm in larvae and we have shown that the levels of SWI/SNF affect the pre-mRNA processing outcome in vivo. Conclusions: We have shown that SWI/SNF can modulate alternative pre-mRNA processing, not only in cultured cells but also in vivo. The effect is restricted to and specific for a subset of transcripts. Our results provide novel insights into the mechanisms by which SWI/SNF regulates transcript diversity and proteomic diversity in higher eukaryotes.},
  language  = {en}
}
@phdthesis{Meduri2017,
  author    = {Meduri, Rajyalakshmi},
  title     = {Elucidation of an intricate surveillance network for cellular U snRNP homeostasis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143173},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Spliceosomal U-rich small ribonucleoprotein particles (U snRNPs) are the major building blocks of the nuclear pre-mRNA splicing machinery. The core composition of U snRNPs includes the name giving U snRNA and a set of seven common (Sm) proteins termed Sm B/B', D1, D2, D3, E, F and G. These Sm proteins are arranged in the form of a toroidal ring on the single stranded conserved sequence element in the snRNA to form the Sm core domain. Even though U snRNPs assemble spontaneously in vitro, their assembly in vivo requires an amazingly large number of trans-acting assembly factors united in the Protein Arginine Methyltransferase 5 (PRMT5) and the Survival Motor Neuron (SMN) complexes. The cytoplasmic assembly pathway of U snRNPs can be divided into the early and the late phase. The early phase is dominated by the assembly chaperone, pICln, a subunit of the PRMT5 complex. This factor binds to Sm proteins and delivers them in a pICln-bound form to the PRMT5 complex. The early assembly phase then segregates into two lines. In one assembly line, a stable hexameric ring intermediate (6S complex) composed of pICln and the five Sm proteins D1, D2, F, E and G, is formed. This intermediate forms at the PRMT5 complex but dissociates from the latter upon completion of its assembly. Within the 6S complex, these Sm proteins are pre-organized into respective spatial positions adopted in the assembled U snRNP. The other assembly line forms a protein trimer composed of pICln, Sm B/B' and D3, which unlike the 6S complex is not released from the PRMT5 complex. As a consequence of their association with pICln, Sm proteins are kinetically trapped and fail to proceed in the assembly pathway. The late phase of the U snRNP formation is dominated by the SMN complex, which resolves this kinetic trap by dissociating pICln from the pre-organized Sm proteins and, subsequently catalyzes the loading of the Sm proteins on the U snRNA. Even though basic principles of U snRNP assembly have been understood in some detail, the question arises as to why cells employ sophisticated assembly machinery for the assembly despite the reaction occurring spontaneously in vitro. A few studies have shown that the system works towards rendering specificity to the assembly reaction. However, Sm proteins in their free form expose hydrophobic surfaces to the cytosolic solvent. Hence, I reasoned that the assembly machinery of snRNPs might also prevent Sm protein aggregation. In this thesis, I describe the work that leads to the discovery of a multi-layered regulatory network for Sm proteins involving post-transcriptional and post-translational surveillance mechanisms. Here, I show that the reduced level of SMN (a key assembly factor of the late phase) leads to the initial tailback of Sm proteins over pICln followed by the transcriptional down regulation of Sm protein encoding mRNAs. In contrast, depletion of pICln, a key factor of the early phase, results in the retention of Sm proteins on the ribosomes followed by their degradation via autophagy. Furthermore, I show that exceeding levels of Sm proteins over pICln caused by overexpression results in aggregation and mis-localization of Sm proteins. Thus, my findings uncover a complex regulatory network that helps to maintain the cellular U snRNP homeostasis by either preventing or clearing the unassembled Sm protein aggregates when they are not faithfully incorporated into the U snRNPs.},
  language  = {en}
}
@article{YinBrocherFischeretal.2011,
  author    = {Yin, Jun and Brocher, Jan and Fischer, Utz and Winkler, Christoph},
  title     = {Mutant Prpf31 causes pre-mRNA splicing defects and rod photoreceptor cell degeneration in a zebrafish model for Retinitis pigmentosa},
  series = {Molecular neurodegeneration},
  volume    = {6},
  journal   = {Molecular neurodegeneration},
  number    = {56},
  doi       = {10.1186/1750-1326-6-56},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-141090},
  pages     = {1-17},
  year      = {2011},
  abstract  = {Background: Retinitis pigmentosa (RP) is an inherited eye disease characterized by the progressive degeneration of rod photoreceptor cells. Mutations in pre-mRNA splicing factors including PRPF31 have been identified as cause for RP, raising the question how mutations in general factors lead to tissue specific defects. Results: We have recently shown that the zebrafish serves as an excellent model allowing the recapitulation of key events of RP. Here we use this model to investigate two pathogenic mutations in PRPF31, SP117 and AD5, causing the autosomal dominant form of RP. We show that SP117 leads to an unstable protein that is mislocalized to the rod cytoplasm. Importantly, its overexpression does not result in photoreceptor degeneration suggesting haploinsufficiency as the underlying cause in human RP patients carrying SP117. In contrast, overexpression of AD5 results in embryonic lethality, which can be rescued by wild-type Prpf31. Transgenic retina-specific expression of AD5 reveals that stable AD5 protein is initially localized in the nucleus but later found in the cytoplasm concurrent with progressing rod outer segment degeneration and apoptosis. Importantly, we show for the first time in vivo that retinal transcripts are wrongly spliced in adult transgenic retinas expressing AD5 and exhibiting increased apoptosis in rod photoreceptors. Conclusion: Our data suggest that distinct mutations in Prpf31 can lead to photoreceptor degeneration through different mechanisms, by haploinsufficiency or dominant-negative effects. Analyzing the AD5 effects in our animal model in vivo, our data imply that aberrant splicing of distinct retinal transcripts contributes to the observed retina defects.},
  language  = {en}
}
@phdthesis{Carstensen2018,
  author    = {Carstensen, Anne Carola},
  title     = {Identification of novel N-MYC interacting proteins reveals N-MYC interaction with TFIIIC},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143658},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {N-MYC is a member of the human MYC proto-oncogene family, which comprises three transcription factors (C-, N- and L-MYC) that function in multiple biological processes. Deregulated expression of MYC proteins is linked to tumour initiation, maintenance and progression. For example, a large fraction of neuroblastoma displays high N-MYC levels due to an amplification of the N-MYC encoding gene. MYCN-amplified neuroblastoma depend on high N-MYC protein levels, which are maintained by Aurora-A kinase. Aurora-A interaction with N-MYC interferes with degradation of N-MYC via the E3 ubiquitin ligase SCFFBXW7. However, the underlying mechanism of Aurora-A-mediated stabilisation of N-MYC remains to be elucidated. To identify novel N-MYC interacting proteins, which could be involved in N-MYC stabilisation by Aurora-A, a proteomic analysis of purified N-MYC protein complexes was conducted. Since two alanine mutations in MBI of N-MYC, T58A and S62A (N-MYC mut), disable Aurora-A-mediated stabilisation of N-MYC, N-MYC protein complexes from cells expressing either N-MYC wt or mut were analysed. Proteomic analysis revealed that N-MYC interacts with two deubiquitinating enzymes, USP7 and USP11, which catalyse the removal of ubiquitin chains from target proteins, preventing recognition by the proteasome and subsequent degradation. Although N-MYC interaction with USP7 and USP11 was confirmed in subsequent immunoprecipitation experiments, neither USP7, nor USP11 was shown to be involved in the regulation of N-MYC stability. Besides USP7/11, proteomic analyses identified numerous additional N-MYC interacting proteins that were not described to interact with MYC transcription factors previously. Interestingly, many of the identified N-MYC interaction partners displayed a preference for the interaction with N-MYC wt, suggesting a MBI-dependent interaction. Among these were several proteins, which are involved in three-dimensional organisation of chromatin domains and transcriptional elongation by POL II. Not only the interaction of N-MYC with proteins functioning in elongation, such as the DSIF component SPT5 and the PAF1C components CDC73 and CTR9, was validated in immunoprecipitation experiments, but also with the POL III transcription factor TFIIIC and topoisomerases TOP2A/B. ChIP-sequencing analysis of N-MYC and TFIIIC subunit 5 (TFIIIC5) revealed a large number of joint binding sites in POL II promoters and intergenic regions, which are characterised by the presence of a specific motif that is highly similar to the CTCF motif. Additionally, N-MYC was shown to interact with the ring-shaped cohesin complex that is known to bind to CTCF motifs and to assist the insulator protein CTCF. Importantly, individual ChIP experiments demonstrated that N-MYC, TFIIIC5 and cohesin subunit RAD21 occupy joint binding sites comprising a CTCF motif. Collectively, the results indicate that N-MYC functions in two biological processes that have not been linked to MYC biology previously. Furthermore, the identification of joint binding sites of N-MYC, TFIIIC and cohesin and the confirmation of their interaction with each other suggests a novel function of MYC transcription factors in three-dimensional organisation of chromatin.},
  subject      = {Biologie},
  language  = {en}
}
@article{HaddadChenZhangetal.2011,
  author    = {Haddad, Dana and Chen, Nanhai G. and Zhang, Qian and Chen, Chun-Hao and Yu, Yong A. and Gonzalez, Lorena and Carpenter, Susanne G. and Carson, Joshua and Au, Joyce and Mittra, Arjun and Gonen, Mithat and Zanzonico, Pat B. and Fong, Yuman and Szalay, Aladar A.},
  title     = {Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus},
  series = {Journal of Translational Medicine},
  volume    = {9},
  journal   = {Journal of Translational Medicine},
  number    = {36},
  doi       = {10.1186/1479-5876-9-36},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-140847},
  pages     = {1-14},
  year      = {2011},
  abstract  = {Introduction: Oncolytic viruses show promise for treating cancer. However, to assess therapeutic efficacy and potential toxicity, a noninvasive imaging modality is needed. This study aimed to determine if insertion of the human sodium iodide symporter (hNIS) cDNA as a marker for non-invasive imaging of virotherapy alters the replication and oncolytic capability of a novel vaccinia virus, GLV-1h153. Methods: GLV-1h153 was modified from parental vaccinia virus GLV-1h68 to carry hNIS via homologous recombination. GLV-1h153 was tested against human pancreatic cancer cell line PANC-1 for replication via viral plaque assays and flow cytometry. Expression and transportation of hNIS in infected cells was evaluated using Westernblot and immunofluorescence. Intracellular uptake of radioiodide was assessed using radiouptake assays. Viral cytotoxicity and tumor regression of treated PANC-1tumor xenografts in nude mice was also determined. Finally, tumor radiouptake in xenografts was assessed via positron emission tomography (PET) utilizing carrier-free (124)I radiotracer. Results: GLV-1h153 infected, replicated within, and killed PANC-1 cells as efficiently as GLV-1h68. GLV-1h153 provided dose-dependent levels of hNIS expression in infected cells. Immunofluorescence detected transport of the protein to the cell membrane prior to cell lysis, enhancing hNIS-specific radiouptake (P < 0.001). In vivo, GLV-1h153 was as safe and effective as GLV-1h68 in regressing pancreatic cancer xenografts (P < 0.001). Finally, intratumoral injection of GLV-1h153 facilitated imaging of virus replication in tumors via (124)I-PET. Conclusion: Insertion of the hNIS gene does not hinder replication or oncolytic capability of GLV-1h153, rendering this novel virus a promising new candidate for the noninvasive imaging and tracking of oncolytic viral therapy.},
  language  = {en}
}
@phdthesis{Klemm2020,
  author    = {Klemm, Theresa Antonia},
  title     = {Minor differences cause major effects: How differential oligomerization regulates the activities of USP25 and USP28},
  doi       = {10.25972/OPUS-19108},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-191080},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Deubiquitinases are regulators of the ubiquitin proteasome system that counteract the ubiquitination cascade by removing ubiquitin from substrates and cleaving ubiquitin chains. Due to their involvment in various important pathways, they are associated with several diseases and may thus present promising drug targets. The two related ubiquitin specific proteases USP25 and USP28 share a highly conserved amino acid sequence but perform distinct biological functions. USP28 plays roles in cell cycle regulation and was also linked to several types of cancer. It adopts oncogenic functions by rescuing the oncoproteins MYC and JUN from proteasomal degradation, which is induced by the E3-ligase SCF (FBW7). Opposingly, USP28 also regulates the stability of the tumor suppressor FBW7 itself. USP25 contributes to a balanced innate immune system by stabilizing TRAF3 and TRAF6 and lately was found to promote Wnt-signaling by deubiquitinating TNKS. Due to the high level of identity of both proteases, a recent attempt to inhibit USP28 led to cross reactivity against USP25. In our study, we characterized both USP25 and USP28 structurally and functionally using x-ray crystallography, biochemical as well as biophysical approaches to determine similarities and differences that can be exploited for the development of specific inhibitors. The crystal structure of the USP28 catalytic domain revealed a cherry-couple like dimer that mediates self-association by an inserted helical subdomain, the USP25/28 catalytic domain inserted domain (UCID). In USP25, the UCID leads to formation of a tetramer composed of two interlinked USP28-like dimers. Structural and functional analysis revealed that the dimeric USP28 is active, whereas the tetrameric USP25 is auto inhibited. Disruption of the tetramer by a cancer-associated mutation or a deletion-variant activates USP25 through dimer formation in in vitro assays and leads to an increased stability of TNKS in cell studies. Furthermore, in vitro data showed that neither ubiquitin nor substrate binding led to the activation of the USP25 tetramer construct. With the structure of the C-terminal domain of USP25, we determined the last unknown region in the enzyme as a separately folded domain that mediates substrate interactions. Combined the structures of the USP25 and USP28 catalytic domains and the functional characterization of both enzymes provide novel insights into the regulation of USPs by oligomerization. Furthermore, we identified individual features of each protease that might be explored for the development of specific small molecule inhibitors.},
  subject      = {Oligomerisation},
  language  = {en}
}
@article{KilincEhrigPessianetal.2016,
  author    = {Kilinc, Mehmet Okyay and Ehrig, Klaas and Pessian, Maysam and Minev, Boris R. and Szalay, Aladar A.},
  title     = {Colonization of xenograft tumors by oncolytic vaccinia virus (VACV) results in enhanced tumor killing due to the involvement of myeloid cells},
  series = {Journal of Translational Medicine},
  volume    = {14},
  journal   = {Journal of Translational Medicine},
  number    = {340},
  doi       = {10.1186/s12967-016-1096-1},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-168914},
  year      = {2016},
  abstract  = {Background The mechanisms by which vaccinia virus (VACV) interacts with the innate immune components are complex and involve different mechanisms. iNOS-mediated NO production by myeloid cells is one of the central antiviral mechanisms and this study aims to investigate specifically whether iNOS-mediated NO production by myeloid cells, is involved in tumor eradication following the virus treatment. Methods Human colon adenocarcinoma (HCT-116) xenograft tumors were infected by VACV. Infiltration of iNOS\(^{+}\) myeloid cell population into the tumor, and virus titer was monitored following the treatment. Single-cell suspensions were stained for qualitative and quantitative flow analysis. The effect of different myeloid cell subsets on tumor growth and colonization were investigated by depletion studies. Finally, in vitro culture experiments were carried out to study NO production and tumor cell killing. Student's t test was used for comparison between groups in all of the experiments. Results Infection of human colon adenocarcinoma (HCT-116) xenograft tumors by VACV has led to recruitment of many CD11b\(^{+}\) ly6G\(^{+}\) myeloid-derived suppressor cells (MDSCs), with enhanced iNOS expression in the tumors, and to an increased intratumoral virus titer between days 7 and 10 post-VACV therapy. In parallel, both single and multiple rounds of iNOS-producing cell depletions caused very rapid tumor growth within the same period after virus injection, indicating that VACV-induced iNOS\(^{+}\) MDSCs could be an important antitumor effector component. A continuous blockade of iNOS by its specific inhibitor, L-NIL, showed similar tumor growth enhancement 7-10 days post-infection. Finally, spleen-derived iNOS+ MDSCs isolated from virus-injected tumor bearing mice produced higher amounts of NO and effectively killed HCT-116 cells in in vitro transwell experiments. Conclusions We initially hypothesized that NO could be one of the factors that limits active spreading of the virus in the cancerous tissue. In contrast to our initial hypothesis, we observed that PMN-MDSCs were the main producer of NO through iNOS and NO provided a beneficial antitumor effect, The results strongly support an important novel role for VACV infection in the tumor microenvironment. VACV convert tumor-promoting MDSCs into tumor-killing cells by inducing higher NO production.},
  language  = {en}
}
@phdthesis{Schoenwetter2021,
  author    = {Sch{\"o}nwetter, Elisabeth Sofie},
  title     = {Towards an understanding of the intricate interaction network of TFIIH},
  doi       = {10.25972/OPUS-16892},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-168926},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The integrity of its DNA is fundamental for every living cell. However, DNA is constantly threatened by exogenous and endogenous damaging agents that can cause a variety of different DNA lesions. The severe consequences of an accumulation of DNA lesions are reflected in cancerogenesis and aging. Several DNA repair mechanisms ensure the repair of DNA lesions and thus maintain DNA integrity. One of these DNA repair mechanisms is nucleotide excision repair (NER), which is famous for its ability to address a large variety of structurally unrelated DNA lesions. A key component of eukaryotic NER is the transcription factor II H (TFIIH) complex, which is not only essential for DNA repair but also for transcription. The TFIIH complex is composed of ten subunits. How these subunits work together during NER to unwind the DNA around the lesion is, however, not yet fully understood. High-resolution structural data and biochemical insights into the function of every subunit are thus indispensable to understand the functional networks within TFIIH. The importance of an intact TFIIH complex is reflected in the severe consequences of patient mutations in the TFIIH subunits XPB, XPD or p8 leading to the hallmark diseases xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Defects in the NER pathway are further associated with several types of cancer including skin cancer. The herein described work focused on five TFIIH subunits derived from the thermophilic fungus Chaetomium thermophilum, the p34/p44 pair and the ternary XPB/p52/p8 complex. The interaction between p34 and p44 was characterized based on a high-resolution structure of the p34_vWA/p44_RING minimal complex. Biochemical studies of the p34/p44 interaction led to the disclosure of an additional interaction between the p34 and p44 subunits, which had not been characterized so far. The p34/p44 interaction was shown to be central to TFIIH, which justifies the presence of several redundant interfaces to safeguard the interaction between the two proteins and might explain why so far, no patient mutations in these subunits have been identified. The p52 subunit of TFIIH was known to be crucial to stimulate the ATPase activity of XPB, which is required during NER. This work presents the first entire atomic resolution structural characterization of p52, which was derived of several crystal structures of p52 variants and a p52/p8 variant thereby demonstrating the interaction between p52 and p8. The precise structural model of p52 offered the possibility to investigate interactions with other TFIIH subunits in more detail. The middle domain 2 of p52 and the N-terminal domain of XPB were shown to mediate the main interaction between the two subunits. An analysis of the p52 crystal structures within recently published cryo-electron microscopy structures of TFIIH provides a model of how p52 and p8 stimulate the ATPase activity of XPB, which is essential for NER and transcription. The structural and biochemical findings of this work provide an additional building block towards the uncovering of the architecture and function of this essential transcription factor.},
  subject      = {DNS-Reparatur},
  language  = {en}
}
@phdthesis{Brosi2021,
  author    = {Brosi, Cornelia},
  title     = {Functional characterization of the TTF complex and its role in neurodevelopmental disorders},
  doi       = {10.25972/OPUS-15778},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-157783},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  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.},
  subject      = {Messenger-RNP},
  language  = {en}
}