Andersen, D.S.; (2008) Characterisation of four Drosophila proteins involved in regulation of gene expression. Doctoral thesis , University of London.

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Abstract

This thesis concerns the characterisation of four Drosophila proteins that are involved in different steps of gene expression. The first protein, dMCRS2, is a nuclear Forkhead associated (FHA) domain protein. dMCRS2 is an essential protein required for growth during development. Drosophila and human MCRS2/MSP58 was previously reported to form a complex with the MOF histone acetyl transferase. My data reveal that in addition to MOF, dMCRS2 can be purified in complex with multiple RNAP II-associated proteins including Cdkll/Pitslre. Moreover, dMCRS2 co-localises with RNAP II complexes on polytene chromosomes in vivo. Thus, dMCRS2 might provide a physical link between the chromatin remodelling MOF complex and RNAP II via its ability to associate with Pitslre. Interestingly, the interaction between dMCRS2 and Pitslre seems to be regulated in response to apoptotic stimuli. The second and third proteins characterised here correspond to the Drosophila homologues of Prp38p (dPrp38), a tri-snRNP splicing factor, and Microfibril-Associated Protein 1 (dMFAPl), a previously uncharacterised protein found in some human spliceosomal fractions. I show here that dMFAPl binds directly to dPrp38 and is required for pre-mRNA processing. dMFAPl, like dPrp38, is essential for growth during development, and my in vivo data show that cells with reduced levels of dMFAPl or dPrp38 proliferate more slowly than control cells and undergo apoptosis. Consistent with this, dsRNA-mediated depletion of dPrp38 or dMFAPl causes cells to arrest in G2/M, and this is paralleled by a reduction in mRNA levels of the mitotic phosphatase string/cdc25. Interestingly dsRNA-mediated depletion of a wide range of core splicing factors elicits a similar phenotype, suggesting that the observed G2/M arrest might be a general consequence of interfering with spliceosome function. The fourth protein, Pixie, can be isolated from cells in complex with eukaryotic initiation factor (elF) 3 and small ribosomal proteins and is required for the assembly of translation initiation competent ribosomes. Thus, depletion of Pixie results in the assembly of empty ribosomes lacking mRNAs and reduced levels of translation initiation. Moreover, Pixie associates with the 40S subunit in an ATP-dependent manner. These data are consistent with Pixie playing a catalytic role in the assembly of complexes required for translation initiation.

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