Untersuchung der molekularen Mechanismen und des Zusammenhanges von TTP-vermitteltem mRNA-Abbau und Translationsunterdrückung mittels strukturellen und biochemischen Methoden
Biochemie
Strukturbiologie
Zusammenfassung der Projektergebnisse
The RNA-binding protein TTP is a member of the tristetraprolin family of factors that bind AU-rich elements (AREs) within the 3’-untranslated region (3’-UTR) of messenger RNA. It is a key factor in regulating inflammation as well as tumor growth and metastasis in mammals. Binding of TTP to ARE-mRNA downregulates their expression through a combination of different molecular mechanisms. This project investigated the interaction of TTP with cellular mRNA decay factors and factors mediating translation repression, using a structural and a biochemical approach. The protein TTP has a central tandem zinc finger domain that is flanked by long intrinsically disordered regions (IDRs). These IDRs are known as activation domains for their ability to activate decay of the bound ARE-mRNA. Work from several groups showed that the IDRs act as interaction platforms for mRNA decay enzymes, providing a mechanistic basis for ARE-mediated decay. However, most of these studies focus on the interaction of TTP with the CCR4-NOT de-adenylation complex, and do not address the other TTP-mediated interactions that regulate expression of ARE-mRNA. In the first project, we investigated the interaction of TTP with the decapping enzyme DCP2. We demonstrated that the N-terminal IDR of TTP directly engages the C-terminal disordered tail of DCP2, leading to stabilization of the proteolytically-sensitive TTP protein in vitro. The TTP-DCP2 interaction is unusual in that it involves the interaction between two IDRs and, despite being a low-affinity interaction, has a significant effect on the stability of ARE-mRNA in cells. Despite the presence of two long IDRs, TTP does not undergo phase-separation on its own. Our studies show that binding of TTP to the C-terminal tail of DCP2 causes it to undergo liquid-liquid phase-separation in vitro. This effect would serve to increase local concentrations of the two proteins and facilitate their interaction. We suggest that molecular crowding in cells could mediate the interaction of TTP and DCP2 and result in the assembly of a decapping complex on a TTP-bound ARE-mRNA. Our work highlights the importance of weak protein-protein interactions in building cellular networks to achieve a final functional outcome. The second phase of the project focused on investigation of the role of TTP in translation repression. To this end, we used structural and biochemical methods to delineate the interaction of TTP with GIGYF2 (GRB10-interacting GYF protein 2), a regulator of mRNA stability and translation. The protein GIGYF2 serves as a protein-protein interaction scaffold by recognizing proline-rich sequences (PRS) on its interaction partners via its conserved GYF domain. Known interaction partners of GIGYF2 include TTP and the TNRC6A/C proteins that are involved in microRNA regulation. GIGYF2 also interacts with the eIF4E-homogue 4EHP and functions as an adaptor between 4EHP and mRNA. The GIGYF2-4EHP axis represses translation of target mRNA without actively degrading them. We systematically analyzed the PRS-motifs of TTP using biochemical and biophysical methods and found that only the first of the three PRS-motifs binds the GYF domain of GIGYF2 with high affinity. We determined the high-resolution X-ray crystal structure of the GIGYF2-GYF domain bound to TTP-PRS1 and identified additional stretches of residues, beyond the known GYF-binding consensus motif, that contribute to binding affinity and specificity. Molecular dynamics simulations show that amino acids flanking the consensus binding motif contribute to the flexibility of the PRS motif, which allows TTP to occupy the binding pocket on the GYF domain of GIGYF2. These in vitro observations are further supported by affinity-purification mass-spectrometry studies in mammalian cells, which show that perturbation of the first PRS motif of TTP disrupts its interaction with GIGYF2 and indirectly, 4EHP. Our studies underscore how consensus motifs within different proteins (such as TTP and TNRC6A/C) have evolved to fine-tune interactions with the same scaffolding factor (GIGYF2) to achieve distinct functional outcomes. Overall, this project has furthered our understanding of the interaction network mediated by TTP to stringently regulate the expression of the highly labile ARE-mRNA transcripts and thereby prevent cellular pathologies.
Projektbezogene Publikationen (Auswahl)
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A conserved structural element in the RNA helicase UPF1 regulates its catalytic activity in an isoform-specific manner. Nucleic Acids Research, 46(5), 2648-2659.
Gowravaram, Manjeera; Bonneau, Fabien; Kanaan, Joanne; Maciej, Vincent D; Fiorini, Francesca; Raj, Saurabh; Croquette, Vincent; Le Hir, Hervé & Chakrabarti, Sutapa
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Intrinsically disordered regions of tristetraprolin and DCP2 directly interact to mediate decay of ARE-mRNA. Nucleic Acids Research, 50(18), 10665-10679.
Maciej, Vincent D; Mateva, Nevena; Schwarz, Juliane; Dittmers, Theresa; Mallick, Megha; Urlaub, Henning & Chakrabarti, Sutapa
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Modulation of RNA-binding properties of the RNA helicase UPF1 by its activator UPF2. RNA, 29(2), 178-187.
Xue, Guangpu; Maciej, Vincent D.; Machado de Amorim, Alexandrina; Pak, Melis; Jayachandran, Uma & Chakrabarti, Sutapa
