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Defects in messenger RNA processing and biogenesis of RNA polymerases contribute to eukaryotic genome instability

University of British Columbia

Genome instability has been observed in mutants involved in various aspects of transcription and RNA processing. The prevalence of this mechanism among essential chromosome instability (CIN) genes remains unclear. In this thesis, it is shown that RNA biogenesis mutants exhibit elevated sensitivity to DNA damaging agents. A secondary screen for increased Rad52 foci in CIN mutants, representing ~25% of essential genes, identified seven essential subunits of the mRNA cleavage and polyadenylation (mCP) machinery. Genome-wide analysis of fragile sites by ChIP-chip of phosphorylated-H2A in these mutants supported a transcription-dependent mechanism of DNA damage characteristic of RNA:DNA hybrids known as R-loops, which were subsequently observed in mCP mutants. Among the CIN mutants with elevated Rad52 foci levels were the GPN proteins, a poorly-characterized and deeply evolutionarily conserved family of three paralogous small GTPases, Gpn1, 2 and 3. The founding member, GPN1/NPA3/XAB1, is proposed to function in nuclear import of RNA polymerase II along with a recently described protein called Iwr1. Here, it is shown that the previously uncharacterized protein Gpn2 binds both Gpn3 and Npa3/Gpn1, and that temperature-sensitive alleles of Saccharomyces cerevisiae GPN2 and GPN3 exhibit genetic interactions with RNA polymerase II mutants, hypersensitivity to transcription inhibition and defects in RNA polymerase II nuclear localization. Importantly, previously unrecognized RNA polymerase III localization defects were observed in GPN2, GPN3 and IWR1 mutant backgrounds but no localization defects of unrelated nuclear proteins or of RNA polymerase I were found. In this study, it was shown that the nuclear import defect of iwr1Δ, but not the GPN2 or GPN3 mutant defects, is partially suppressed by fusion of a nuclear localization signal to the RNA polymerase II subunit Rpb3. These data, combined with strong genetic interactions between GPN2 and IWR1 suggest that the GPN proteins function upstream of Iwr1 in RNA polymerase II and III biogenesis. We propose that the three GPN proteins execute a common function in RNA polymerase assembly and subsequent transport. These findings demonstrate how mRNA cleavage and polyadenylation and proper RNA polymerase assembly contribute to maintenance of genome integrity and may be relevant to certain human cancers.

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2013-04-16

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/44216

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/