Research Collection Doctoral Thesis Structural and Functional Studies of mRNA Stability Regulators Author(s): Ripin, Nina Publication Date: 2018-11 Permanent Link: https://doi.org/10.3929/ethz-b-000303696 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH NO. 25327 Structural and functional studies of mRNA stability regulators A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH ZÜRICH (Dr. sc. ETH Zürich) presented by NINA RIPIN Diplom-Biochemikerin, Goethe University, Frankfurt, Germany Born on 06.08.1986 citizen of Germany accepted on the recommendation of Prof. Dr. Frédéric Allain Prof. Dr. Stefanie Jonas Prof. Dr. Michael Sattler Prof. Dr. Witold Filipowicz 2018 “Success consists of going from failure to failure without loss of enthusiasm.” Winston Churchill Summary Posttranscriptional gene regulation (PTGR) is the process by which every step of the life cycle of an mRNA following transcription – maturation, transport, translation, subcellular localization and decay - is tightly regulated. This is accomplished by a complex network of multiple RNA binding proteins (RNPs) binding to several specific mRNA elements. Such cis-acting elements are or can be found within the 5’ cap, the 5’ untranslated region (UTR), the open reading frame (ORF), the 3’UTR and the poly(A) tail at the 3’ end of the mRNA. Adenylate-uridylate-rich elements (AU-rich elements; AREs) are heavily investigated regulatory cis- acting elements within 3’untranslated regions (3’UTRs). These are found in short-lived mRNAs and function as a signal for rapid degradation. AREs are present in 5-8% of human genes involved in the regulation of many essential cellular processes, such as stress response, cell cycle regulation and apoptosis and must therefore be tightly regulated. In the cytoplasm, trans-acting ARE binding proteins regulate the transport localization, stability and translation of these mRNAs. One of these factors is the embryonic lethal abnormal visual (ELAV)/ Human antigen R (HuR) protein. It increases the stability and/ or the translation of many important cellular mRNAs. Another cis-activing element is the poly(A) tail of mRNA, which protects the mRNAs from degradation. These are bound by multifunctional poly(A)-binding proteins (PABPs), which play a central role in translation initiation, translation termination and mRNA decay. In this study, we have investigated the mRNA stability regulators HuR and PABPC1, both containing multiple RNA recognition motifs (RRMs). Excitingly, some single RRMs have several functions due to the presence of additional binding interfaces that allow them to bind both RNA and other factors. We characterized the C-terminal RRM of HuR, which is hypothesized to be involved in RNA binding, homo-dimerization and protein-protein interacting. We show the first 1.9- Å-resolution crystal structure of HuR RRM3 bound to several short ARE-motifs. Our structure reveals the presence of the homodimer. The combination of several biophysical techniques validate the homo-dimerization and promiscuous RNA binding in solution. Additionally, the binding of the canonical AUUUA pentameric motifs, found in the majority of AREs, is possible by the recognition of two registers. Excitingly, RRM3 homo-dimerization increases the affinity for RNA, highlighting the cooperativity between the two binding surfaces. Moreover, despite the known stabilizing role of HuR, we provide evidence that RRM3 counteracts this effect in a Huh7 cell - based ARE reporter assay containing multiple AUUUA motifs. Finally, we investigated the mechanism of the cytoplasmic PABP RRM1 in binding to poly(A) and to the anti-proliferative B- cell translocation gene (BTG2) protein. BTG2 recruits the CCR4-associated factor 1 (CAF1), a subunit of CCR4-NOT deadenylase complex, to induce deadenylation of mRNAs. We show that PABPC1 RRM1 uses its α1 to bind BTG2 while simultaneously binding the poly(A) RNA. This interaction seems to orient the poly(A) 3’end such that it is close to the CAF1 enzymatic pocket. Our findings provide new details of the HuR RRM3-RNA recognition and homo-dimerization as well as the PABPC1 RRM1-poly(A)-BTG2 binding to recruit CAF1 and thus highlight the diversity of RRMs. Zusammenfassung Posttranskriptionelle Genregulation (PTGR) ist ein Prozess nach der Transkription, bei dem jeder Schritt des Lebenszyklus einer mRNA - Prozessierung, Transport, Translation, subzelluläre Lokalisierung und Abbau - streng reguliert wird. Dieser wird durch ein komplexes Netzwerk von RNA-bindenden Proteinen (RBP) erreicht, die mehrere spezifische mRNA-Elemente binden. Solche cis-wirkenden Elemente sind oder liegen innerhalb der 5'-Kappe, der 5'-untranslatierten Region (UTR), des offenen Leserahmens (ORF), des 3'UTR und des Poly(A) -Schwanzes am 3'- Ende. Adenylat-Uridylat-reiche Elemente (AU-reiche Elemente; AREs) sind stark untersuchte regulatorische cis-wirkende Elemente innerhalb von den 3'-untranslatierten Regionen (3'UTRs). Diese finden sich in kurzlebigen mRNAs und fungieren als ein Signal für den schnellen mRNA Abbau. AREs sind in 5-8% der menschlichen Gene vorhanden, die an der Regulation vieler essenzieller zellulärer Prozesse beteiligt sind, wie Stressreaktion, Zellzyklusregulation und Apoptose und müssen daher streng überwacht werden. Im Zytoplasma, trans-wirkende ARE- bindende Proteine steuern die Lokalisierung, Stabilität und Translation dieser mRNAs. Einer dieser Proteine ist das „embryonic lethal abnormal visual (ELAV)/ human antigen R (HuR)“ Protein. Es erhöht die Stabilität und/ oder Translation vieler dieser zellulärer mRNAs. Ein weiteres cis-wirkendes Element ist der Poly(A) -Schwanz von mRNAs, der die mRNAs vor Abbau schützt. Dieser wird durch multifunktionelle Poly(A) -bindenden Proteine (PABPs) gebunden, die eine zentrale Rolle bei der Translationsinitiation, Translationstermination und dem mRNA-Zerfall spielen. Das Ziel dieser Doktorarbeit ist die Untersuchung der mRNA bindenden Proteine HuR und PABPC1. Diese enthalten mehrere RNA-bindinge Domänen/ „RNA recognition motifs, (RRMs)“. Interessanterweise haben einige dieser RRMs zahlreiche Funktionen aufgrund der Anwesenheit von mehreren Bindungsstellen. Diese ermöglichen sowohl RNA- als auch Protein-Bindung. Wir charakterisierten die C-terminale RRM von HuR, von welcher angenommen wird, dass sie an RNA-Bindung, Homodimerisierung und Protein-Protein-Wechselwirkung beteiligt ist. Wir zeigen die erste 1,9-Å-Kristallstruktur von HuR RRM3, die an mehrere kurze ARE-Motive gebunden ist. Unsere Struktur zeigt die Anwesenheit eines Homodimers. Durch die Kombination mehrerer biophysikalischer Metoden validieren wir die Homodimerisierung und die RNA-Bindung in Lösung. Darüber hinaus ist die Bindung der kanonischen AUUUA-Pentamer-Motive, die in den meisten AREs gefunden werden, durch die Erkennung von zwei Bindungsregistern möglich. Die RRM3-Homodimerisierung erhöht die Affinität für RNA und verdeutlicht die Kooperativität zwischen den beiden Bindungsoberflächen. Schließlich, trotz der bekannten stabilisierenden Rolle von HuR, liefern wir Beweise, dass RRM3 diesem Effekt in einem auf Huh7-Zellen-basierten ARE-Reporter-Test entgegenwirkt. Darüber hinaus untersuchten wir den Mechanismus der zytoplasmatischen PABP-RRM1 bei der Bindung an poly(A) und an das anti-proliferative B-Zell- Translokationsgen (BTG2) -Protein. BTG2 rekrutiert den CCR4-assoziierten Faktor 1 (CAF1), eine Untereinheit des CCR4-NOT-Deadenylase-Komplexes, um die Deadenylierung von mRNAs zu induzieren. Wir zeigen, dass PABPC1 RRM1 die α1 verwendet, um BTG2 zu binden, während es gleichzeitig die poly(A) RNA bindet. Diese Wechselwirkung scheint das Poly(A) 3'-Ende so auszurichten, dass es nahe der CAF1-enzymatischen aktivem Zentrums liegt. Unsere Ergebnisse liefern interessante Informationen über die HuR-RRM3-RNA-Erkennung und Homodimerisierung sowie die PABPC1-RRM1-BTG2-Bindung, um CAF1 zu rekrutieren und verdeutlichen damit die Diversität von RRMs. Acknowledgment My time during the doctorate was the toughest challenge I ever faced. I grew as a scientist but also as a person. For that, a special thanks to Fred. You took me in under special circumstances and for that, I am very grateful. The time in your group was and still is one of the best and valuable experiences I had in my life. Thank you for all your support. I would like to acknowledge Prof. Stefanie Jonas, Prof. Michael Sattler and Prof. Witold Filipowicz for joining as co-referees for my thesis. Michael, I really enjoyed our discussions and your challenging questions in Parpan. Another very big thanks goes to Malgosia. You have been always there for me. Your wisdom is highly precious and I learned a lot from you. Fred D, thanks a lot for teaching me how to set up and the basics of NMR experiments and especially for correcting my English whenever it was needed and thanks to Julien for introducing me to ITC, your support on the HuR project and all the discussions we had. Nana and Irene, it was fun working with you. One cannot imagine better colleagues and friends in the lab. Ahmed, you are always there to help and support others. Thank you for being such a good listener. Yaro, thanks for the fun working evenings and late discussions. Thea, Gerry, Fred D, Alvar and Simon, thank you for keeping our NMR spectrometers running. Stefanie, many thanks for all your advice on the cell culture experiments and more.