Investigation of transcriptional and translational regulation of the Neurospora crassa alternative oxidase

• Author / Creator

  Bosnjak, Natasa

• Mitochondria are organelles found in most eukaryotes and supply the cell with the majority of its energy needs in the form of ATP. Most of the proteins needed for mitochondrial function are nuclear encoded. Therefore, mitochondria communicate their functional status to the nucleus, resulting in the modulation of nuclear gene expression. In the event of a disruption of the standard mitochondrial electron transport chain, the nuclear-encoded aod-1 gene is induced in Neurospora crassa. Aod-1 encodes an alternative oxidase (AOX) that provides a branch point off the standard chain. When present, AOX transfers electrons from coenzyme Q directly to oxygen.
  The induction of AOX is dependent on two transcription factors in N. crassa: AOD2 and AOD5. Previous ChIP-seq analysis identified several other genes bound by these transcription factors. The most robust binding peak was found to be in the promoter of NCU06940, a gene encoding a hypothetical protein. Using qPCR analysis on strains lacking AOD2 or AOD5, I demonstrated that the expression of NCU06940 was dependent on these proteins. I hypothesized that NCU06940 had a role in stress because transcripts were upregulated in the presence of an AOX inducer. I examined the growth of a strain in which the NCU06940 gene had been deleted. I found that the growth of the strain on various carbon sources, at different temperatures, and in the presence of inhibitors of mitochondrial function was indistinguishable from wild-type. Thus, the function of the gene remains unknown.
  In N. crassa, the AOX is encoded by the aod-1 gene. In normal (non-inducing) conditions, aod-1 is transcribed at very low levels and no protein can be detected. A strain of N. crassa that carries mutations in the tyrosinase gene (T) was previously found to produce relatively high amounts of aod-1 transcript in non-inducing conditions with no detectable AOD1 protein. Growth of the strain in inducing conditions results in an increase in transcription and production of the protein. I was able to rule out the tyrosinase gene as a regulator of aod-1 transcription using qPCR analysis for aod-1 transcript levels by examining the progeny of a cross between a wild-type strain and the tyrosinase mutant strain. Furthermore, I analyzed whole genome sequencing data on the parents and selected progeny strains and identified the flbA gene and the kin-9 genes mutated in only the high aod-1 transcript expressing progeny and parental strains. Further analysis strongly suggested that the mutated flbA gene was responsible for the elevated transcripts.
  I also attempted to determine why non-induced cells with high levels of aod-1 transcript do not contain AOD1 protein in their mitochondria. I showed that the protein does not accumulate in the cytosol due to lack of import into mitochondria. I also showed that it is likely not synthesized and rapidly degraded. Thus, the aod-1 mRNA may be subjected to translational control in non-inducing conditions.

• Subjects / Keywords

  • Neurospora
  • Mitochondria
  • Genetics
  • Biology

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3X63BN22

• License

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