CHOP, or Gadd153, is a 29 kDa protein that plays a pivotal role in the mediation of cellular stress-induced cell death. The expression of the gene encoding CHOP/Gadd153 is regulated at both the transcriptional and post-transcriptional levels. Compared to transcription, the regulation of Chop gene expression at the post-transcriptional level is much less understood. In this study, the role played by the 3’-untranslated region of CHOP mRNA (3UTRChop) in mediating mRNA stability was examined. A reporter plasmid was constructed so that the mRNA expressed has 3UTRChop as its 3’-untranslated region. Partial 5’ deletions, or deletion of short internal (~30 nucleotides) sequences, or the deletion of a putative AU-rich element (ARE) within 3UTRChop, all resulted in the elevation of the steady state levels of mRNA and the encoded reporter protein, EGFP. Deletion of the ARE or sequences remote from ARE resulted in the reduced rate of mRNA degradation. Such data suggested that 3UTRChop is closely related to mRNA stability. The effect of cellular stress on the functioning of 3UTRChop was studied by examining the change in mRNA level in cells treated with arsenic trioxide (ATO). The presence of arsenic stress stimulated a marked increase in the steady state level of not only the reporter mRNA, but also control mRNAs that did not have 3UTRChop as the 3’-untranslated region. A non-specific effect of arsenic stress on mRNA levels was therefore suggested. Consistent with the increase in the level of reporter mRNA, the expression of EGFP protein was also increased. Arsenic stress and partial deletion of 3UTRChop produce additive increase in mRNA level and EGFP protein level implying that the mRNA destabilizing function of 3UTRChop is unlikely to be stress-regulated. The contribution of 3UTRChop in mRNA translation was then examined using reporter constructs that expressed EGFP mRNA having its original 5’ and 3’-untranslated regions replaced with 5UTRChop and 3UTRChop respectively. In the presence of wild-type 3UTRChop, the abolition of the translation repressor functions of 5UTRChop produced only mild increase in EGFP expression. However, the additional partial deletion of 3UTRChop resulted in massive increase in EGFP expression. In the presence of wild-type 5UTRChop, the partial deletion of 3UTRChop resulted in only a small increase in EGFP expression. Such data demonstrated a complementary relationship between 5UTRChop and 3UTRChop in the regulation of Chop expression in unstressed cells. EGFP mRNA having 5UTRChop and 3UTRChop as the 5’ and 3’-untranslated region respectively expressed significant EGFP protein only in the presence of ATO. The expression of EGFP was not significantly affected with swopping of 3UTRChop with another 3UTR. 3UTRChop is therefore not essential for the mediation of ATO-stimulated expression of EGFP. The present study demonstrated that full length 3UTRChop may have constitutive mRNA destabilizing effect that is not alleviated by cellular stress. The evaluation of the relative contributions of 5UTRChop and 3UTRChop in mRNA translation suggested a model for Chop gene expression whereby the eventual protein level of Chop is determined by 5UTRChop-mediated translation as well as by 3UTRChop-mediated destabilization of mRNA template. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xii, 187 p.; also includes graphics (some col.) Includes bibliographical references (p. 175-187). Available online via OhioLINK's ETD Center

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xviii, 216 p.; also includes graphics (some col.) Includes bibliographical references (p. 196-216). Available online via OhioLINK's ETD Center

Hannover, University, Diss., 2005.

The propagation of the genetic information into proteins is mediated by messenger- RNA (mRNA) intermediates. In eukaryotes mRNAs are synthesized by RNA- Polymerase II and subjected to translation after various processing steps. Earlier it was suspected that the regulation of gene expression occurs primarily on the level of transcription. In the meantime it became evident that the contribution of post- transcriptional events is at least equally important. Apart from non-coding RNAs and metabolites, this process is in particular controlled by RNA-binding proteins, which assemble on mRNAs in various combinations to establish the so-called “mRNP- code”. In this thesis a so far unknown component of the mRNP-code was identified and characterized. It constitutes a hetero-trimeric complex composed of the Tudor domain-containing protein 3 (TDRD3), the fragile X mental retardation protein (FMRP) and the Topoisomerase III beta (TOP3β) and was termed TTF (TOP3β-TDRD3-FMRP) -complex according to its composition. The presented results also demonstrate that all components of the TTF-complex shuttle between the nucleus and the cytoplasm, but are predominantly located in the latter compartment under steady state conditions. Apart from that, an association of the TTF-complex with fully processed mRNAs, not yet engaged in productive translation, was detected. Hence, the TTF-complex is a component of „early“ mRNPs. The defined recruitment of the TTF-complex to these mRNPs is not based on binding to distinct mRNA sequence-elements in cis, but rather on an interaction with the so-called exon junction complex (EJC), which is loaded onto the mRNA during the process of pre-mRNA splicing. In this context TDRD3 functions as an adapter, linking EJC, FMRP and TOP3β on the mRNP. Moreover, preliminary results suggest that epigenetic marks within gene promoter regions predetermine the transfer of the TTF-complex onto its target mRNAs. Besides, the observation that TOP3β is able to catalytically convert RNA-substrates disclosed potential activities of the TTF-complex in mRNA metabolism. In combination with the already known functions of FMRP, this finding primarily suggests that the TTF-complex controls the translation of bound mRNAs. In addition to its role in mRNA metabolism, the TTF-complex is interesting from a human genetics perspective as well. It was demonstrated in collaboration with researchers from Finland and the US that apart from FMRP, which was previously linked to neurocognitive diseases, also TOP3β is associated with neurodevelopmental disorders. Understanding the function of the TTF-complex in mRNA metabolism might hence provide important insight into the etiology of these diseases. / Die Umwandlung der genetischen Information in Proteine erfolgt über Boten-RNA (mRNA) -Intermediate. Diese werden in Eukaryonten durch die RNA-Polymerase II gebildet und nach diversen Prozessierungs-Schritten der Translationsmaschinerie zugänglich gemacht. Während man früher davon ausging, dass die Genexpression primär auf der Ebene der Transkription reguliert wird, ist heute klar, dass post- transkriptionelle Prozesse einen ebenso wichtigen Beitrag hierzu leisten. Neben nicht-kodierenden RNAs und Metaboliten tragen insbesondere RNA- Bindungsproteine zur Kontrolle dieses Vorgangs bei. Diese finden sich in unterschiedlichen Kombinationen auf den mRNAs zusammen und bilden dadurch den sog. „mRNP-Code“ aus. Im Rahmen dieser Dissertation wurde eine bislang unbekannte Komponente des mRNP-Codes identifiziert und charakterisiert. Es handelt es sich dabei um einen hetero-trimeren Komplex, welcher aus dem Tudor Domänen Protein 3 (TDRD3) dem Fragilen X Mentalen Retardations-Protein (FMRP) sowie der Topoisomerase III beta (TOP3β) besteht. Aufgrund seiner Zusammensetzung wurde dieser TTF (TOP3β-TDRD3-FMRP) -Komplex genannt. In der vorliegenden Arbeit konnte der Nachweis geführt werden, dass sämtliche Komponenten des TTF-Komplexes zwischen Zellkern und Cytoplasma pendeln, unter Normalbedingungen jedoch vornehmlich im Cytoplasma lokalisiert sind. Des Weiteren ließ sich eine Assoziation des TTF-Komplexes mit mRNAs nachweisen, die zwar vollständig prozessiert, jedoch noch nicht Teil der produktiven Phase der Translation sind. Der TTF-Komplex ist somit eine Komponente „früher“ mRNPs. Die Rekrutierung des TTF-Komplexes an definierte mRNPs wird nicht durch Bindung an spezifische mRNA-Sequenzelemente bedingt, sondern basiert auf einer Interaktion mit dem sog. Exon Junction Complex (EJC), welcher im Kontext des pre-mRNA Spleißens auf die mRNA geladen wird. Hierbei spielt TDRD3 als Adapter zwischen dem EJC, FMRP und TOP3β die entscheidende Rolle. Präliminäre Experimente legen darüber hinaus den Schluss nahe, dass epigenetische Markierungen im Promotor-Bereich distinkter Gene von entscheidender Bedeutung für den Transfer des TTF-Komplexes auf dessen Ziel-mRNAs sind. Einen wichtigen ersten Hinweis auf die potentielle Funktion des TTF-Komplexes im Kontext des mRNA Metabolismus erbrachte die Beobachtung, dass TOP3β in der Lage ist RNA katalytisch umzusetzen. Dieser Befund lässt in Verbindung mit den bereits beschriebenen Aktivitäten von FMRP vermuten, dass der TTF-Komplex die Translation gebundener mRNAs kontrolliert. Zusätzlich zu seiner Rolle im mRNA Metabolismus ist der TTF-Komplex auch aus humangenetischer Sicht hoch interessant. So konnte in Zusammenarbeit mit finnischen und US-amerikanischen Forschern gezeigt werden, dass neben FMRP, einem bekannten Krankheitsfaktor neurokognitiver Syndrome, auch TOP3β mit neurologischen Entwicklungsstörungen assoziiert ist. Das Verständnis der Funktion des TTF-Komplexes im mRNA Metabolismus könnte daher wichtige Einblicke in die Etiologie dieser Krankheiten liefern.

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Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 71-76). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references. Available also from UMI/Dissertation Abstracts International.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "May 2001." Typescript. Includes bibliographical references (leaves 109-111). Also available on the Internet.