Biogenesis of ribosomal RNAs in Dictyostelium discoideum

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Ribosomal RNAs (rRNAs) are encoded in the amoeba Dictyostelium discoideum in approximately 100 copies of an extrachromosomal palindrome, which are thought to be derived from a single chromosomal locus. To analyse this molecularly, we will generate a genetically traceable version of this locus and analyse whether or not the marker can later also be found in the extrachromosomal elements. We will determine whether the content of rDNA copies is constant during development or whether new rDNA is being synthesized upon hatching, or during any other time point in development. We further will analyse whether all 100 extrachromosomal elements are actively transcribed, or only a subset. A likely means of transcriptional silencing of a sub-set of these elements is DNA methylation. This is indirectly supported by our observation of a large fraction of endogenous 21mer RNAs with sequence identity to the transcribed region of the rDNA palindrome by deep sequencing. To analyse whether (RNA-mediated) DNA methylation is operating, we will subject DNA from different genetic backgrounds to treatment with methylation sensitive restriction enzymes and bisulfite sequencing to map the DNA methylation pattern on the extrachromosomal elements. In the spores of the amoeba, precursors of the mature rRNAs are accumulated. Having detailed earlier the sequences of mature rRNAs and their precursors, we wish to analyse here molecularly, what stops rRNA processing during development, and at what point. We will determine the sequences of the stored precursors and determine their sub-cellular localisation. To this end, we have developed a protocol for RNA fluorescence in situ hybridization (FISH) in D. discoideum, as a complementary tool to a wide range of already established techniques in this lab (cRT-PCR, Northern Blot, primer extension, RT-PCR etc.). Unexpectedly, we could observed that the Dicer-protein DrnB is involved in the maturation of rRNAs and we will detail this phenomenon in this project. We will use a bioinformatics to identify further enzymes that are involved in the rRNA maturation and generate gene deletion strains by using a one-step cloning protocol recently established in this lab. Should these strains not viable, we will use a knock-down approach by RNAi to determine the effects of the respective enzyme on rRNA maturation.