As our knowledge of the genome expands, so does our understanding of the characteristics of what we define as genes. Small Open Reading Frame (smORF) genes have eluded gene annotation until very recently, and evidence is mounting that these very short nucleotide sequences encode functional peptides that are ≤100 amino acids in size. From work conducted in the fruit fly, our lab has successfully characterised three Drosophila smORFs, of which two have been shown to have a function in higher vertebrates, including humans. The functional characterisation of one of these conserved smORF encoded peptides (SEPs), Hemotin, is presented in this thesis. Though the overall number is still low compared to the abundance of potential smORF-encoding genes in Drosophila, the information gathered here allows us to speculate on the wider role of smORF peptides through cell-based imaging studies conducted on Drosophila cells. Here, I will discuss the various techniques that can and should be employed in order to study the functions of SEPs. Chapter III describes the various phenotypic studies conducted on the Hemotin smORF which is expressed in Drosophila haemocytes, and are integral to the fruit fly immune system. This study showed that connecting subcellular localisation of an SEP to a direct functional assay in cells can reveal functional characteristics of the peptide for further study. Chapter IV details the results from a tagging-transfection assay, which began initially as a way to independently corroborate the translation of smORF mRNAs that were assessed as such by Ribosome Profiling. This experiment resulted in the discovery of several mitochondrial-localised SEPs in Drosophila S2 cells, opening the door for the direct functional assay described in Chapter V. The results from a small-scale RNAi screen conducted on the mitochondria of S2 cells provided a reliable read-out for functionality of a large proportion of the smORFs that were screened. This assay can potentially be used as a phenotypic read-out of mitochondrial-SEP function in any cell or tissue type. Elucidation of smORFs and the functions of the peptides that they encode will help us to expand the Drosophila proteome, along with providing evidence of their functionality across every organism in which they are found. Considering that characterised SEPs play very important roles in physiology and health, it is time for smORFs to be acknowledged as the important genomic elements that they are.

Despite the benefits of sexual reproduction, sharing a genome can put constraints on the evolvability of a species. This is due to sexual conflict, where the interests of each sex is in direct opposition to one another, and the benefit of one sex can be the cost of the other sex (i.e. sexual antagonism). Sex chromosomes have been the focus of much of the research done on sexual conflict due to their unique nature and are particularly interesting in the context of sexually antagonistic variance. In the first experiment (Chapter 2), I used experimental evolution to investigate the standing sexually antagonistic variation on the X-chromosome of the common vinegar fly, Drosophila melanogaster. Unlike most other experimental evolution experiments where selection has been limited to males, I limited the inheritance of the X-chromosome to females only. I used a non-recombining Xchromosome balancer to control the inheritance of the female-limited X-chromosome. Throughout the evolution experiment, I tested different phenotypic traits that have previously been shown to be sexual antagonistic, as well as investigating how the transcriptome changed through female-limited selection (Chapter 3). The results were mixed but indicated that limiting selection of the X-chromosome to females could, to some extent, change the antagonistic variation and move traits towards the female optimum. In the second experiment (Chapter 4), I exchanged sex chromosomes between populations with divergent geographic origins. I used flies with special genetic constructs (e.g. autosomal balancers, fused-X chromosomes) to control the population crosses, so that sex chromosomes were introduced into a new background without any prior interaction. I found that introducing a novel sex chromosome increased male reproductive fitness through improved sperm competition at the cost of offspring viability. 25 generations after introducing the novel sex chromosome (Chapter 5), the increase in male fitness was undetectable and their fitness was again the same as the wild types. Collectively, this indicates an antagonistic coevolution between the sex chromosomes. Together, these two experiments shed new light on sexual conflict and the antagonistic coevolution between the sexes at the genetic level, both between and within the sex chromosomes. These novel insights could help further the understanding of how sex chromosomes may affect speciation.

The Hox genes encode a family of developmental regulators that are essential for the normal patterning of the animal body axis. Their correct expression is controlled by a number of mechanisms including RNA processing, a molecular system that allows the formation of alternative mRNAs from a single gene. Previous work in the Alonso Lab has demonstrated that RNA processing of the Drosophila Hox gene Ultrabithorax (Ubx) by means of alternative splicing and alternative polyadenylation plays an important role during Drosophila development, but the mechanisms underlying these regulatory processes are not well understood. In this project we found that the Drosophila neural RNA binding protein ELAV has an important role in the control of both Ubx alternative splicing and polyadenylation during embryonic development. Furthermore, by conducting a series of in vitro experiments we demonstrate that ELAV is able to interact with two specific RNA elements located within Ubx intronic sequences and mutation of such elements abolishes the interaction. We also establish that embryos carrying a loss of function mutation in the elav gene produce lower levels of Ubx mRNA and protein suggesting a role of ELAV on Hox gene expression. Finally we investigated the roles that other Drosophila factors including RNA binding proteins, chromatin regulators and splicing regulatory proteins may have on Ubx RNA processing and found several potential regulators. All in all our work contributes to the understanding of the molecular basis of Hox RNA processing control during Drosophila development.