Post-transcriptional control is one of many layers of regulation of gene expression within the cell. Within this, RNA stability is known to play a critical role in determining the period of time an RNA species is able to elicit its function; such as an mRNA to be translated into protein or a miRNA to bind and repress its targets. The stability of a cytoplasmic RNA is dependent upon its degradation which can occur from either the 5’ end by Pacman or from the 3’ end by a family of related exoribonucleases; the Dis3 family.
The aim of this thesis was to investigate the role 3’-5’ exoribonucleases play in post-transcriptional control of gene expression and how these relate to the development of a model tissue. Drosophila melanogaster have two highly conserved members of the Dis3 family, Dis3 and Dis3L2, both of which have been implicated in human disease. Dis3 has been recurrently mutated in multiple myeloma and acute myeloid leukaemia whilst Dis3L2 has been shown to be mutant in the human overgrowth syndrome, Perlman syndrome.
Due to their role in developmental diseases the function of both enzymes was investigated in the growth and development of the wing imaginal disc. A reverse genetics approach was used by driving RNA interference specifically in the wing imaginal disc which was directed by the highly specific GAL4-UAS system. The results within this thesis show that Dis3 is critical for organism viability together with the correct development of the wing with its loss resulting in large scale apoptosis. Using miRNA-sequencing has allowed the identification of a novel role for Dis3 in the regulation miRNA stability with miR-252-5p presenting as a specific target of Dis3.
Unlike, Dis3, Dis3L2 was shown to be dispensable for organism viability and the development of the adult wing identifying a more specific function. This was identified to be in the control of developmental proliferation. Loss of dis3L2 within the wing imaginal disc resulted in increased proliferation of the wing imaginal discs cells resulting in the overgrowth of the wings and the wing imaginal discs by 20% compared to parental controls. The observed overgrowth was consistent with the human disease of Perlman syndrome which itself is characterised by foetal overgrowth thus identifying a conserved function for Dis3L2 in the control of tissue growth.
RNA sequencing was performed in a global, unbiased, approach in order to identify targets of Dis3L2 which become misregulated following its knockdown and may therefore drive the overgrowth phenotype. This revealed 2 potential targets of Dis3L2, pyrexia and CG2678 which were shown to significantly increase in expression at the post-transcriptional level. Neither of these targets are known to be involved in proliferation, however, further investigation would be required to elucidate the mechanism by which Dis3L2 regulates proliferation within a developing tissue.
The role identified for Dis3L2 in regulating proliferation is in stark contrast to that of the 5’-3’ exoribonuclease Pacman which has previously been shown to regulate apoptosis. Work in this thesis has built upon the relationship between Dis3L2 and Pacman and shown that they indeed function to regulate distinct pathways through the regulation of largely discrete sets of transcripts.
|Date of Award||Jun 2016|