Date

2013

Document Type

Dissertation

Degree

Doctor of Philosophy

Department

Molecular Biology

First Adviser

Ware, Vassie C.

Other advisers/committee members

Iovine, M. Kathryn; Kuchka, Michael R.; Pederson, Thoru

Abstract

Gene duplication is a contributing factor to genome evolution in eukaryotes. With an additional copy, selective pressure is relieved, allowing for accumulation of genetic variation and possible development of new or altered functions. Ribosomal protein (Rp) genes are a common class of duplicated genes found throughout eukaryotes. Typically encoding highly similar or identical proteins at separate loci, duplicated Rps were originally thought to be redundant and to relieve the high demand for translation. However, recent reports in yeast have shown phenotypic differences between Rp paralogue knockouts, suggesting functional non-redundancy. Little effort has been devoted toward elucidating the function of Rp paralogues in eukaryotes other than in yeast. Furthermore, in yeast, paralogous Rps are typically highly identical, making studying gene function difficult without protein tagging. To explore whether duplicated Rp genes have redundant roles, we focused on the eukaryotic-specific RpL22e family in Drosophila melanogaster. The Drosophila RpL22e family consists of two members, the ancestral rpL22e and its duplicate rpL22e-like, which are 37% identical. Divergence is evident in the genomic sequence, codon usage, and protein sequence, but whether this results in novel functions has not been previously addressed and is the focus of this dissertation.It is widely known that the ancestral RpL22e is ubiquitous, but our data show that RpL22e-like expression is primarily restricted to the male germline and is a true ribosomal component. Further investigation shows that in testis tissue, RpL22e is primarily SUMOylated and phosphorylated. Only unmodified RpL22e co-sediments with the translation machinery in Drosophila S2 cells, leading to the interpretation that the majority of testis RpL22e is not part of the translation machinery and that paralogue functions are non-redundant. Immunohistochemical analysis further supports non-redundant paralogue roles, as RpL22e is primarily restricted to the nucleoplasm in the maturing meiotic germline; RpL22e-like is cytoplasmic in these cells. Additionally, there is an unequal requirement for RpL22e members in vivo, as only rpL22e is essential in the fly.Taking the data in this dissertation together, it is evident that the Drosophila RpL22e paralogues have diverged in function within the male germline. RpL22e assumes an additional and unique role compared to RpL22e-like.

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