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Transfer RNA nucleotidyltransferases (CCA-adding enzymes) are important enzymes, which catalyze the attachment of a CCA triplet to the 3‘ end of tRNAs, an essential requirement for subsequent aminoacylation. These special enzymes function in a fascinating manner without a nucleic acid template. Furthermore, a substrate affinity switch from CTP to ATP is fulfilled with high specificity and the reaction is precisely terminated after addition of the terminal ATP. In some bacteria, the CCA-adding activity is divided into two enzymes: a CC- and an A-adding enzyme. This diversity that was long only assigned to Bacteria. However, the growing number of eukaryotic genomes allowed for deep bioinformatic investigation, revealing several eukaryotic organisms with an unusual amount of tRNA nucleotidyltransferase genes. In the present work, the function of several tRNA nucleotidyltransferases found in the genome of certain fungi, amoeba and choanoflagellates was investigated. For the tRNA nucleotidyltrans-ferases detected in Salpingoeca rosetta and Schizosaccharomyces pombe, a divided activity similar to bacterial CC- and A-adding enzymes could be observed. Additionally, in the amoeba Dictyostelium discoideum two bona fide CCA-adding enzymes were found, which are inversely regulated during the developmental cycle. In the amoeba Acanthamoeba castellanii, four different tRNA nucleotidyltransferases with different activities, localization and evolutionary origin were identified. Moreover, a method for the precise analysis of mature tRNAs by high-throughput sequencing was established as well. This method includes the specific ligation of a hairpin adapter molecule, which complementarily and highly efficiently binds to tRNAs with a 3’-CCA end resulting in a very specific preparation of tRNAs for high-throughput sequencing. It also allows for analysis of some modified bases usually found in tRNAs, which was used to analyze the alteration of certain tRNA modifications during the developmental cycle of D. discoideum.:Erklärung der Selbstständigkeit II List of Abbreviations V Bibliografische Darstellung VII Zusammenfassung 1 Summary 6 Chapter I 11 1.1. Transfer RNAs 12 1.1.1. Structure and maturation of transfer ribonucleic acids (tRNAs) 12 1.1.2. tRNAs as regulatory molecules and their role in diseases 13 1.1.3. Sequencing of tRNAs – a special challenge 14 1.1.4. The 3’-CCA end of tRNAs 15 1.2. tRNA nucleotidyltransferases 16 1.2.1. Classification and biological roles 16 1.2.2. Class II tRNA nucleotidyltransferases 18 1.2.3. Enzymes with split activity – bacterial CC- and A-adding enzymes 19 1.2.4. Two types of eukaryotic tRNA nucleotidyltransferases 21 1.2.5. Distribution of eukaryotic organisms with multiple tRNA nucleotidyltransferase genes 22 1.3. Aim of the work 23 1.4. References 25 Chapter II 33 Chapter III 44 Chapter IV 75 Chapter V 100 Chapter VI 119 Publications and Presentations IX Author Contribution Statement XI Danksagung XVI

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [119]-139).

RNA interference or RNAi was first discovered as an experimental approach that induces potent sequence-specific gene silencing. Remarkably, subsequent studies on dissecting the molecular mechanism of the RNAi pathway reveal that RNAi is conserved in most eukaryotes. In addition, genes and mechanisms related to RNAi are employed to elicit the regulation of endogenous gene expression that controls a variety of important biological processes. To investigate the mechanism of RNAi in the nematode C. elegans, we performed genetic screens in search of RNAi deficient mutants (rde). Here I report the summary of the genetic screens in search of rde mutants as well as the identification of two novel genes required for the RNAi pathway, rde-3 and rde-8. In addition, we demonstrate that some of the rde genes, when mutated, render the animals developmentally defective, suggesting that these rde genes also function in developmental gene regulation. This work presents novel insights on the components of the RNAi pathway and the requirement of these components in the regulation of endogenous gene expression.