Molecular and Developmental Analysis of Non-Coding RNA Metabolism in C. elegans: the Exoribonuclease XRN2 and the RNA- Binding Proteins SART-3 and USIP-1 Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Stefan Rüegger Aus Maur, Schweiz Basel, 2014 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Dieses Werk ist unter dem Vertrag „Creative Commons Namensnennung-Keine kommerzielle Nutzung- Keine Bearbeitung 3.0 Schweiz“ (CC BY-NC-ND 3.0 CH) lizenziert. Die vollständige Lizenz kann unter creativecommons.org/licenses/by-nc-nd/3.0/ch/ eingesehen werden. Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Nancy Hynes, Dr. Helge Grosshans, Dr. Javier Martinez Basel, den 18.02.2014 Prof. Dr. Jörg Schibler (Dekan) Namensnennung-Keine kommerzielle Nutzung-Keine Bearbeitung 3.0 Schweiz (CC BY-NC-ND 3.0 CH) Sie dürfen: Teilen — den Inhalt kopieren, verbreiten und zugänglich machen Unter den folgenden Bedingungen: Namensnennung — Sie müssen den Namen des Autors/Rechteinhabers in der von ihm festgelegten Weise nennen. Keine kommerzielle Nutzung — Sie dürfen diesen Inhalt nicht für kommerzielle Zwecke nutzen. Keine Bearbeitung erlaubt — Sie dürfen diesen Inhalt nicht bearbeiten, abwandeln oder in anderer Weise verändern. Wobei gilt: Verzichtserklärung — Jede der vorgenannten Bedingungen kann aufgehoben werden, sofern Sie die ausdrückliche Einwilligung des Rechteinhabers dazu erhalten. Public Domain (gemeinfreie oder nicht-schützbare Inhalte) — Soweit das Werk, der Inhalt oder irgendein Teil davon zur Public Domain der jeweiligen Rechtsordnung gehört, wird dieser Status von der Lizenz in keiner Weise berührt. Sonstige Rechte — Die Lizenz hat keinerlei Einfluss auf die folgenden Rechte: o Die Rechte, die jedermann wegen der Schranken des Urheberrechts oder aufgrund gesetzlicher Erlaubnisse zustehen (in einigen Ländern als grundsätzliche Doktrin des fair use bekannt); o Die Persönlichkeitsrechte des Urhebers; o Rechte anderer Personen, entweder am Lizenzgegenstand selber oder bezüglich seiner Verwendung, zum Beispiel für Werbung oder Privatsphärenschutz. Hinweis — Bei jeder Nutzung oder Verbreitung müssen Sie anderen alle Lizenzbedingungen mitteilen, die für diesen Inhalt gelten. Am einfachsten ist es, an entsprechender Stelle einen Link auf diese Seite einzubinden. Quelle: http://creativecommons.org/licenses/by-nc-nd/3.0/ch/ Datum: 12.11.2013 Acknowledgments I would like to express my deepest gratitude to my PhD advisor, Helge Großhans, for his patient, caring, and motivating guidance. I would like to express my sincere gratitude to Iskra Katic for always being willing to share her immense knowledge of C. elegans, for providing cutting-edge worm tools, and for invaluable hands-on help with worm injections. I wish to thank Daniel Hess for performing mass spectrometry experiments, which crucially contribute to the work at hand. I am thankful for the scientific support from my thesis committee, Nancy Hynes and Javier Martinez. I wish to express my gratitude to Boehringer Ingelheim Fonds for financial support but particularly also for personal support and for organizing many enjoyable events. 2 Contents Acknowledgments ......................................................................................................................................... 2 1 Summary ............................................................................................................................................... 4 2 RNA metabolism in eukaryotes ............................................................................................................. 6 3 The exoribonuclease XRN2 in C. elegans: Its role in development and its interplay with the novel protein PAXT-1 ............................................................................................................................................ 11 3.1 Abstract and contributions ......................................................................................................... 12 3.2 Introduction ................................................................................................................................ 13 3.2.1 XRN 5’-to-3’ exoribonucleases ............................................................................................ 13 3.2.2 The nuclear exoribonuclease XRN2 .................................................................................... 14 3.2.3 The cytoplasmic exoribonuclease XRN1 ............................................................................. 17 3.2.4 Publication: “MicroRNA turnover: when, how, and why” .................................................. 19 3.3 Results and Discussion ................................................................................................................ 31 3.3.1 Publication: “Engineering of a Conditional Allele Reveals Multiple Roles of XRN2 in Caenorhabditis elegans Development and Substrate Specificity in MicroRNA Turnover” ................ 32 3.3.2 Additional results ................................................................................................................ 55 3.4 Outlook ....................................................................................................................................... 60 4 USIP-1 is a terminal transferase acting upstream of SART-3 in spliceosome assembly ..................... 61 4.1 Abstract ....................................................................................................................................... 62 4.2 Introduction ................................................................................................................................ 63 4.3 Results ......................................................................................................................................... 72 4.4 Discussion .................................................................................................................................... 83 4.5 Supplementary figures ................................................................................................................ 87 4.6 Methods ...................................................................................................................................... 91 5 Conclusion ........................................................................................................................................... 95 6 Publication: “PAXT-1 promotes XRN2 activity by stabilizing it through a conserved domain” .......... 96 7 Publication: “The Decapping Scavenger Enzyme DCS-1 Controls MicroRNA Levels in Caenorhabditis elegans” .................................................................................................................................................... 107 8 References ........................................................................................................................................ 115 9 Curriculum vitae ................................................................................................................................ 126 3 1 Summary About three quarters of a eukaryotic genome are transcribed into RNA. However, only <2% of these transcripts are translated into protein while the bulk of transcripts execute their biological function as RNA. Non-protein coding RNAs (ncRNAs) associate with proteins in ribonucleoprotein particles (RNPs) to regulate gene expression at various stages thereby greatly increasing the functional complexity of the genome. Nonetheless, the function and mode of action of the vast majority of ncRNAs is unknown and even in well studied examples little is known about the post-transcriptional regulation of ncRNAs themselves. In the thesis at hand, I explored the molecular and developmental functions of proteins implicated in the metabolism of ncRNAs, namely the miRNA-degrading enzyme XRN2 and the U6 snRNA- interacting proteins SART-3 and USIP-1. The XRN2 project was a collaboration with Takashi Miki and Hannes Richter. XRN2 project XRN2 is a conserved 5’-to-3’ exoribonuclease involved in various pathways including transcription termination and processing of precursor forms of rRNAs and snoRNAs. Our lab had established a function of XRN2 in the turnover of mature miRNAs, however, whether XRN2 targets all or specific miRNAs in vivo remained unclear. Although XRN2 substrates have extensively been characterized, the developmental function of XRN2 is essentially unexplored. Moreover, knowledge of co-factors regulating XRN2 function beyond transcription termination is scarce in multicellular organisms. In order to elucidate the developmental role of XRN2, we characterized an xrn-2 null and xrn-2 temperature- sensitive mutant. We found that XRN2 is essential during several stages of C. elegans development, including embryogenesis, and that only specific miRNAs are affected by XRN2 in vivo. Co- immunoprecipitations identified PAXT-1 (PArtner of XRN-Two 1) as a tight interaction partner of XRN2. paxt-1 depletion enhanced the xrn-2ts mutant phenotype and a paxt-1 null mutant slowed-down miRNA degradation in vivo, similar to XRN2 inactivation. These observations, as we showed, are due to a stabilizing effect of PAXT-1 on XRN2. Truncation mutants of PAXT-1 revealed a conserved N-terminal domain of unknown function, DUF3469, sufficient for XRN2 binding. We were excited to discover