Janis Melanie Kruse

• Dicer and Drosha proteins are eukaryotic endoribonucleases that belong to the highly conserved RNase III family. Their prominent function is to cleave double stranded RNA into small RNA duplexes with 20-25 bp in size that comprise typically 2 nt 3´-overhangs. These characteristic small RNAs are recognized by cellular Argonaute proteins and mediate, by virtue of complementary base pairing with the target, posttranscriptional gene silencing, epigenetic modifications or silencing of invasive RNA such as viruses. The best studied types of such regulatory small RNAs, micro RNAs and small interfering RNAs (miRNAs and siRNAs, respectively) were also identified in the unicellular model organism Dictyostelium discoideum. The genome of this amoeba encodes for two Dicer-like proteins referred to as DrnA and DrnB that are potentially involved in the biogenesis of small RNA. However, little is known about their general properties and functions in such cellular pathways. In this work it has been shown that the two Dicer-like proteins have despite their great similarity in domain structure different intracellular localizations that resemble plant Dicing- and animal P-bodies, respectively. The internal binding domain for double stranded RNA (dsRBD) of DrnB was further demonstrated to be not essential in miRNA processing in vivo. Instead DrnB was shown to co-localize with the dsRBD containing protein dsRBP_B, indicating that the function of DrnB in this pathway is rather established by a trans-interacting than by its internal dsRBD. Polyclonal antibodies against DrnA were raised in this work, and revealed an even expression of DrnA throughout the development of D. discoideum into a multicellular fruiting body. This antibody recognized DrnA also in a large protein complex as shown by native PAGE approaches and allowed to co-purify putative protein interaction partners. A presumably essential role of DrnA for the amoeba´s viability prevented possibly its chromosomal deletion. It was demonstrated in this study that the gene re-integrated into the genome by an unknown mechanism which appears to be based on in vivo ligation of knockout constructs and re-recombination of the gene. In a further part of the study on DrnA and DrnB, their RNase III domains were investigated with respect to their catalytic properties and substrate specificity in vitro. By the use of different RNA substrates it was shown that the recombinant domains can cleave one strand in dsRNA molecules that comprise regions of mismatches. Perfectly base paired dsRNA or single stranded RNA, however, were not processed. This unexpectedly restricted substrate recognition contrasts the findings in experiments with Dicer in other organisms, and offers new insights into the properties of RNase III domain containing proteins which appear to be divergent in Dictyostelium. Finally, preliminary investigations of ribosomal RNA in the drnB knockout strain revealed an accumulation of small RNAs that are derived from the 17S rRNA and its precursor. This observation suggests that DrnB participates in rRNA related pathways; however its detailed contribution awaits further examination. In summary, Dicer-like proteins in Dictyostelium seem to combine attributes from Dicer and Drosha in plants, animals and yeast: They form nuclear bodies like they do in plants, but they also assemble into cytoplasmic structures like in animals, and they finally appear to be involved in ribosomal RNA pathways like RNase III proteins in yeast. From an evolutionary perspective this reflects the phylogenetic position of the Dictyostelium lineage that branched out after plants but before animals and fungi, and may place DrnA and DrnB to an intermediate stage in the evolution of Dicer and Drosha in higher eukaryotes.