Waltenspiel, Bernhard (2011) Characterisation of the gene deflated, the Drosophila melanogaster orthologue of human Integrator subunit 7. PhD thesis, James Cook University.

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Abstract

Transcription of the majority of spliceosomal RNAs, small nuclear RNAs (snRNAs), as well as all protein-coding genes is accomplished by RNA polymerase II (RNAPII). Recently, a novel protein complex called 'Integrator' was identified in human cells that associates with the C-terminal domain (CTD) of RNAPII’s largest subunit. Integrator has previously been shown to be critical for the co-transcriptional 3' end processing of snRNAs, as depletion or mutation of at least two of the twelve known Integrator subunits causes 3' end misprocessed snRNAs to accumulate. Genes encoding Integrator subunits are well conserved in metazoa, including Drosophila, but are absent from the genomes of unicellular organisms. Previous studies of the Drosophila orthologue of Integrator subunit 7 (Ints7), deflated, associated deflated with cellular roles that are difficult to reconcile with a function in snRNA 3' end processing. These include pleiotropic defects in deflated mutants throughout the entire development indicative of functions in cell proliferation and/or cell signalling. This thesis explores the functional conservation of deflated as a subunit of the Drosophila Integrator complex and examines the possibility of deflated being multifunctional.

In an attempt to address the involvement of deflated in snRNA biogenesis as well as other cellular processes, a series of studies were conducted using Drosophila for experimentation. Changes in snRNA 3' end processing were analysed via quantitative real-time PCR (qPCR) in a mutant deflated background. In comparison to heterozygous control larvae, homozygous second instar defl^L larvae accumulate significant amounts of 3' end misprocessed snRNA precursors and also display defects in pre-mRNA splicing of some, but not all, mRNA transcripts. This is consistent with deflated functioning as an Ints7 Integrator subunit and Integrator having a critical role in snRNA 3' end processing in Drosophila.

A functional involvement of deflated as a subunit of the Drosophila Integrator complex was further explored using the yeast two-hybrid system (Y2H). Deflated was found to bind to two of the twelve known Integrator subunits in Drosophila as well as to the DSS1 protein that initiated discovery of the Integrator complex (in human cells) but does not play a role in snRNA biogenesis. To explore the possibility that deflated is multifunctional, as indicated by previous findings in Drosophila, a commercial Y2H screen was commissioned to identify non-Integrator binding partners of Deflated. Two novel physical interactors of Deflated were identified and confirmed in this study via pairwise Y2H assays. The first was the Mcm2 subunit of the MCM2-7 replicative helicase and the second the dynein light chain Dlc90F of cytoplasmic dynein. These observations support a multifunctional role of deflated.

In the final set of experiments, Deflated localization studies during Drosophila development were undertaken using a custom made anti-Deflated antibody. Immunofluorescence confocal microscopy revealed that Deflated is ubiquitously expressed in the nuclei of early Drosophila embryos but becomes more and more restricted in its tissue distribution with ongoing development. Anti-Deflated staining first follows sites of rapid cell proliferation and is only seen in the developing CNS and epithelia of mainly ectodermal origin from gastrulation until late larval stages. In adults, Deflated's localization was analysed in the male reproductive system, where it strongly stained primary spermatocytes, and in female ovaries, where it was found to stain all egg chamber follicle cells until mid-oogenesis. Later in oogenesis, anti-Deflated staining becomes restricted mainly to the anterior follicle cell population and a small proportion of follicle cells at the posterior pole. Using cDNA rescued homozygous defl^L females to analyse egg chamber development in a mutant background, defects in the posterior follicle cell epithelium became apparent after mid-oogenesis. These data establish a clear link between the epithelial specific expression of Deflated and a function in epithelial organisation. An additional finding of the anti-Deflated staining was colocalization of Deflated with y-tubulin to centrosomes in follicle cells. This localization may well reflect the identified Y2H physical interaction between Deflated and Dlc90F.

Together, these data suggest that deflated encodes a multifunctional protein that is required for correct snRNA 3' end formation as well as other cellular processes. The findings of this study indicate possible roles of Deflated at centrosomes, in epithelial organisation and, in association with Mcm2, in either DNA replication or transcription of protein-coding genes. Further research of deflated and other Integrator subunits will be required to reveal the full extent of functions beyond Integrator's role in snRNA 3' end processing and why Integrator complex subunits are restricted to only metazoan species.

Item ID:	18799
Item Type:	Thesis (PhD)
Keywords:	Drosophila, genetics, RNA, genetic mutation, Integrator subunits, gene transcription, Integrator subunit 7, metazoan genetics, snRNA biogenesis, cell proliferation, Drosophila melanogaster, orthologue of human Integrator subunit 7, 3'end processing,mRNA , messenger RNA, protein-coding genes, complex proteins, gene expression, multifunctional genes
Date Deposited:	28 Nov 2011 23:13
FoR Codes:	06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060103 Cell Development, Proliferation and Death @ 50% 06 BIOLOGICAL SCIENCES > 0604 Genetics > 060405 Gene Expression (incl Microarray and other genome-wide approaches) @ 50%
SEO Codes:	97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 100%
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