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Abstract

Centromeric chromatin of metazoa is highly repetitive and cannot be classified as typically euchromatic or heterochromatic. Centromeric regions harbour few to no genes but they are transcribed into non-coding RNAs in many species studied to date. In Drosophila melanogaster, one of the transcribed (peri)centromeric sequences is the 359 bp satellite III (sat III) repeat. Sat III RNA transcripts localize to the (peri)centromere, where they stabilize the newly incorporated centromeric histone CENP-A (or CID in Drosophila). When sat III RNA levels are reduced, cells harbour mitotic defects, such as lagging chromosomes and micronuclei formation. Although sat III has an important role at the centromere, little is known about sat III RNA regulation and its interaction partners, as well as possible other functions. In this thesis, I set out to identify new sat III RNA-associated proteins in a sat III RNA pulldown. Among the identified interacting proteins was a hitherto uncharacterized complex of four unknown proteins, which proved to be important for the Drosophila germline development. We, therefore, named it Centromeric Transcript-Associated Gonadal (Centagon) complex. I show that the Centagon complex proteins interact with each other as well as with sat III RNA. The sat III-interacting Centagon complex is expressed in the nucleoli of germ cells and somatic cells in the ovary and its knockdown results in severe germline defects. Depletion of Centagon components by RNAi resulted in defects in the ovary germarium including a loss of germ cells resulting in rudimentary ovaries and agametic flies. Immunofluorescent stainings with a germ stem cell marker, suggest that the defect may be caused by a failure of the germ cells to properly differentiate. On the other hand, depletion in the ovary egg chambers resulted in a developmental arrest of the egg chambers and nurse cell chromatin aberrations. Importantly, sat III RNA levels were upregulated up to 50-fold in the Centagon depleted ovaries, indicating that the Centagon complex is involved in sat III RNA repression. Furthermore, my experiments show that the egg chamber developmental arrest and chromatin aberrations depend on high sat III transcript levels, since I could partially rescue the phenotype by reducing sat III RNA levels. Knockdown experiments in somatic ovary cells and the male germ line showed that the Centagon complex is also essential in other cell types and not ovary-specific. In conclusion, I identified a new sat III RNA-associated protein complex that is involved in sat III RNA regulation and showed that sat III RNA has a negative effect on ovary development when upregulated.