Jan Diesend

• The amoeba Dictyostelium discoideum is a versatile and genetically tractable model organism used in research for many cellular processess. This work is focused on the biochemical study of two different processes in the amoeba. The first part was the establishment of monitoring techniques for viral infections in the amoeba. For this purpose, two detection method should be established: a qPCR-based assay for the counting of the Acanthamoeba polyphaga mimivirus (APMV) genomes and it was observed that no net increase occurred in D. discoideum AX2 cells. APMV is taken up into the amoeba, however, the number of viral particles decreased until 6 hours post infection (hpi), indicating that APMV was not able to enter the replicative stage, possibly failing in the phagolysosomal pathway. Infection of phagolysosomal mutants indicated that lysosomal enzyme might be key in the defense against APMV. The second part covers the potential ribosome heterogeneity during development of D. discoideum with a focus on the 2´-O-methylation (2´-O-Me) and pseudouridylation (Ψ). Both chemical modifications are introduced sequence-specifically by box C/D and box H/ACA small nucleolar RNPs (snoRNPs), respectively. Using bioinformatics and RNAseq analysis, 30 novel box C/D snoRNAs were identified, however, in silico approaches failed to deliver box H/ACA snoRNA candidates. Modified nucleotides were determined by the application of RiboMeth-seq for 2´-O-Me and HydraPsiSeq for Ψ. Several 2´-O-Me and Ψ sites were found to be substoichiometrically modified and some positions displayed dynamic modification levels during the development of the amoeba. Experiments and previous data on the U3 snoRNA implied a sudden generation of novel and potentially specialized ribosomes during development. Taken together, the presented data from D. discoideum is the first evidence for ribosome heterogeneity in the Amoebozoa supergroup, allowing to suggest that it is a common feature of all eukaryotes.