The Role of the ORB2 RNA-binding Protein in Post-transcriptional Regulation

RNA-binding proteins (RBPs) are key components of the post-transcriptional regulatory framework and can determine the translational fate of their RNA targets. A wide variety of post-transcriptional regulatory mechanisms are employed during the early Drosophila embryo and it has been established as a system well suited for the study of these mechanisms. In Drosophila, ORB2 is an RBP expressed in the early embryo. It has been shown to regulate translation of bound mRNAs, but relatively little is known about what RNAs it targets in early embryogenesis and the mechanisms by which it confers this regulation. In this thesis, I show that ORB2 associates with over 450 genes in the early embryo. Analysis of these genes shows that they are translationally repressed during the first two hours of development and are degraded during the maternal to zygotic transition (MZT). Using a dual luciferase assay, I show that ORB2 confers translational repression on a target mRNA and I map this repressive function to the C-terminal ZZ domain of the ORB2 protein. Next, I identify over 200 proteins which interact with ORB2 in the early embryo. This list of interactors includes protein components of the 43S preinitiation complex of translation initiation and components of the Cup repressive complex. I found that the ZZ domain is necessary for ORB2’s association with both complexes. Finally, I show that deletion of the ZZ domain of ORB2 causes major defects during the process of spermatogenesis. In these mutants, the localization of protein gradients to the apical tip of spermatid cysts is disrupted and the process of spermatid individualization fails to complete. These defects ultimately cause male sterility in these mutant flies.

Department dc:contributor.department

Molecular Genetics

Year dc:date.issued

2023

Author dc:creator

• Low, Timothy

Advisor dc:contributor.advisor

• Lipshitz, Howard D

• IP-MS
• ORB2
• RIP-Seq
• RNA-binding
• translation

Handle dc:identifier.uri

https://hdl.handle.net/1807/149812

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/149812