Molecular Function and Regulation of Aub Arginine Methylation in the piRNA Pathway

Citation

Huang, Xiawei (2021) Molecular Function and Regulation of Aub Arginine Methylation in the piRNA Pathway. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/5734-db78. https://resolver.caltech.edu/CaltechTHESIS:05192021-055633048

Abstract

Transposon elements (TEs, Transposons) are DNA sequences that can change their position within the genome. TEs, so-called 'jump genes', sometimes create mutation which will disrupt genes or damage the genome integrity by causing double-stranded DNA breaks and germ cell death. It is important for living animals to maintain the integrity of genetic information during reproduction. In Metazoa germline, cells use the piwi-interacting RNA (piRNA) pathway, which is an RNA – interference (RNAi) based defense strategy to protects the genome from the attacking of the "selfish" transposons. The core unit of the piRNA pathway is the RNA-induced silencing complex (RISC), a conserved family of Argonaute protein that interacts with small (19–33 nt) RNA guides in eukaryotic species. In Drosophila melanogaster, three PIWI-clade Argonaute proteins are present in the germline – Aubergine (Aub), Argonaute 3 (Ago3) and Piwi. PIWI proteins together with their substrate piRNAs forming the RISC to suppress the TE activity. Arginine (Arg) methylation is an important post-translational modification among Argonaute proteins. Defects of Arginine methylation cause the de-repression of deleterious TE.

The work present in this thesis examines the molecular function and regulation mechanism of Aub Arginine methylation in Drosophila germline cells. Chapter I presents a general introduction to the TEs, key components of the piRNA pathway, potential piRNA processing site, "nuage", and the correlation between arginine methylation and its interaction partner, Tudor domain-containing proteins. Chapter II presents the piRNA biogenesis in Drosophila germline and somatic cells, dividing the piRNA pathway into cytoplasmic and nuclear branches. We describe the mechanism of piRNA 5' end and 3' end formations. Chapter III explores the specific molecular function of Aub arginine methylation in the piRNA ping-pong cycle. Further, we decipher the regulation mechanism of Aub Arginine methylation, addressing its biological meaning for the piRNA biogenesis. In chapter 4, we developed a heterologous two-hybrid system to identify factors that directly interact with Piwi, which can further be applied to elucidate the interaction network of the piRNA pathway. In chapter 5, we discuss the potential role of phase separation in the assembly of ping-pong processing granule and the biological meaning in the piRNA biogenesis. We also propose the future plan and the protocol to examine the hypothesis in the future.

Thesis Files