Programmed Translational Readthrough in Drosophila melanogaster

by Prajwal Karki
Doctoral thesis
Date of Examination:2019-06-10
Date of issue:2020-06-05
Advisor:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Marina Rodnina
Referee:PD Dr. Halyna Shcherbata
Persistent Address: http://dx.doi.org/10.53846/goediss-8015

 

 

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Abstract

English

Translational readthrough (TR) is extensively used by viruses to expand their limited genome capacity. The pervasiveness of TR across eukaryotic genomes is only recently being explored. Systems biology approaches such as comparative phylogenetics, combined with ribosome profiling studies have led to the identification of several hundred genes that can undergo TR in Drosophila melanogaster. However, only limited number of these genes have been experimentally studied. Due to the lack of systemic biochemical studies, the actual mechanism of TR regulation and the role of regulated TR in proteome expansion in D. melanogaster remains largely unexplored. In this study, we develop a dual luciferase reporter assay system to analyze a set of genes from D. melanogaster that have been phylogenetically predicted to undergo TR. We provide experimental validation and quantification of the extent of TR in these genes and also analyze the impact of immediate stop codon context on TR modulation. For one particular candidate, aPKC, we use systematic mutational analysis to delineate the minimal primary sequence motif responsible for driving efficient TR. Furthermore, in order to understand the biological significance and phenotypic outcome of gene-specific TR, we employ CRISPR/Cas9-based genome editing to create genetic mutants of the large Maf transcription factor, traffic jam (tj), that exhibit constitutive TR or abolished it. We identify tissue-specific regulation of TR in tj, wherein the expression of Tj-TR isoform is restricted to the nervous tissues. The TR extension selectively attenuates the native Tj function, specifically in the determination of morphogenetic behavior and spatial distribution of cap cells in adult ovaries. Conversely, the TR extension positively amplifies the selective gene regulatory function of native Tj in downregulation of the adhesion protein Fasciclin III. Using high-throughput RNA sequencing, we further dissect the role of TR in tj in shaping the transcriptome profile in adult brains. Our results hint towards a complex mode of regulation of Tj function by TR that operates via conditional fine-tuning of its specific gene regulatory functions.
Keywords: Readthrough, Translation, traffic jam, development, Drosophila
 
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