RNA Interactions of the Zebrafish Germ Plasm Organizer Bucky ball

by Hazem Khalifa
Doctoral thesis
Date of Examination:2023-03-16
Date of issue:2023-07-24
Advisor:Prof. Dr. Ernst A. Wimmer
Referee:Prof. Dr. Ernst A. Wimmer
Referee:Prof. Dr. Rolando Rivera-Pomar
Referee:PD Dr. Roland Dosch
Sponsor:DAAD
Persistent Address: http://dx.doi.org/10.53846/goediss-9831

 

 

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Abstract

English

Reproduction is the fundamental feature of all living organisms, ensuring their survival and continuance. For their reproduction, multicellular organisms develop highly specialized cells, termed germ cells. In many animals, germ cells are specified upon receiving cytoplasmic determinants, termed germ plasm (Gp). These determinants are inherited maternally through the oocyte and are exclusively maintained by the future germ cells during early embryogenesis. The zebrafish protein Bucky ball (Buc) is the first discovered Gp organizer in vertebrates, as it controls Gp assembly and germ cell formation. Buc functionally mimics the Drosophila protein Oskar (Osk), a known Gp organizer in invertebrates. On the biochemical level, Osk interacts with the Drosophila Vasa protein and nanos mRNA, and both interactions are required for the formation of germ cells in Drosophila. Although Buc is a novel protein, which has no known functional domains, it was recently shown to interact with the zebrafish Vasa protein, mimicking a biochemical activity of Osk in Drosophila, and suggesting that Buc interacts also with RNAs during the process of germ cell specification in zebrafish. Analyzing the effect of buc mutation on the RNA composition during oogenesis and embryogenesis, I serendipitously detected that buc mRNA is specifically maintained and potentially translated in germ cells, which requires the last 25 nucleotides of its 3’ untranslated region (3’UTR), thereby demonstrating that the buc 3’UTR can be used to generate germ cell markers. Moreover, I show that Buc interacts with zebrafish nanos3 (nos3) mRNA both in a cell-free system as well as in vivo. These results demonstrate for the first time that Buc acts as an RNA binding protein. Unexpectedly, I found that Buc binds nos3 mRNA independently of the 3’UTR. Furthermore, systematic protein truncation experiments indicate that nos3 mRNA binding by Buc requires the middle of the Buc protein. For further investigations of the biochemical and biophysical behaviors of the Buc protein, I managed for the first time to express the recombinant Buc protein in a highly soluble form.
Keywords: Bucky ball; germ plasm; germline; nanos3; zebrafish; primordial germ cells; RNA-binding proteins; oogenesis; embryogenesis; RNA posttranscriptional regulation