Vorschau

PDF, Englisch - Hauptdokument Download (23MB) | Lizenz: Rechte vorbehalten - Freier Zugang

Abstract

Every cell division relies on functional centromeres to guarantee correct chromosome segregation. They are particularly important in the germline to ensure successful reproduction and embryonic development. Centromeres are epigenetically defined by the incorporation of the histone H3 variant CENP-A and are embedded into pericentromeric chromatin. Non-coding RNAs generated by transcription from centromeric and pericentromeric regions propagate centromere function and specification. Yet, little is known about how (peri-) centromeric RNAs are regulated and which interaction partners they have.

The primary goal of my doctoral thesis was to identify factors that are associated with and influence the function of the lncRNA SatIII from the (peri-) centromeric regions of the X chromosome of Drosophila melanogaster. In this thesis, I identified RNA-binding proteins (RBPs) present at the centromere by re-analyzing a published mass spectrometry dataset that was produced by a crosslinked CENP-A pulldown approach. In doing so, I found out that the RBP Fmr1 is one of the top enriched factors and demonstrated that it binds to SatIII RNA in vitro. Additionally, I performed Fmr1 CLIP-Seq and showed that Fmr1 binds to (peri-)centromeric RNAs in the female germline. Moreover, Fmr1 associates with a subset of mRNAs including some that encode proteins which are important for centromere function, and also positively affects their translation. In order to find novel interactors of Fmr1 mass spectrometry was used. Thereby, I was able to identify the RBP Rump, which interacts with Fmr1 in an RNA-dependent manner, indicating their co-binding to common target RNAs. In fact, there is an overlap between Fmr1 and Rump CLIPSeq data in the non-coding RNAs that both proteins target. Additionally, I produced RNA-Seq data and showed that both proteins regulate the levels of non-coding RNAs that emerge from the centromere and elsewhere in the genome (in the male germline). Finally, Rump was shown to be required in maintaining CENP-A levels in mature sperm.

Overall, this thesis demonstrates an implication of the RBPs Fmr1 and Rump in centromere function and regulation in the germline of Drosophila melanogaster.