Saccharomyces cerevisiae has the ability to alter its growth based on how much of a particular carbon source is present in the environment; when glucose is depleted the cells will enter the post-diauxic phase. Based on previous evidence, we suspect that mRNA that encode for mitochondrial proteins are degraded by a novel autophagic decay pathway in the post-diauxic phase, requiring the ribonucleases Xrn1 and Rny1 for proper mitochondrial growth. First, we tested the localization of the ribonucleases Xrn1 and Rny1 to determine if their localization changes due to stressful conditions, and if these results can indicate a change in their function. Second, to test if an autophagic mRNA decay pathway exists, we used the MS2 labeling technique to fluorescently tag specific mRNAs within the cell in order to see if they associate with structures that maybe involved in autophagic mRNA decay, such as autophagosomes and stress granules. We tested three specific mRNAs for their localization: PGK1, DPI8 and COX5A. PGK1 encodes for a protein kinase that aids in metabolism, while DPI8 and COX5A both encode for mitochondrial proteins. We were able to determine that we can visualize mRNA using the MS2-CP-GFP fusion protein, however we believe that the MS2-CP-mCherry fusion did not detect MS2-tagged mRNAs. Using the MS2-CP-GFP assay, we were able to determine that PGK1 localizes to mitochondria in the post-diauxic phase.
Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control mechanism for the dynamic regulation of gene expression. NMD degrades transcripts containing a premature termination codon (PTC) more than 50-55 nucleotides upstream of the final exon-exon junction. Although NMD is a ubiquitous mechanism for degrading RNA transcripts in all eukaryotes, there is great variety in the efficiency and specificity of the degradation mechanism. While most transcripts containing a PTC are degraded via NMD, transcripts containing a PTC can evade NMD and produce truncated or full-length proteins1. NMD efficiency may also vary based on gene sequence, intracellular location, tissue, or on an individual level. This study aimed to aid the understanding of NMD as an endogenous control for gene expression by evaluating NMD efficiency in homogenous cell cultures. We evaluated NMD efficiencies in human embryonic kidney cells by transfecting cell cultures with dual-fluorescing reporters for NMD. We measured fluorescent levels through flow cytometry, and surprisingly detected varying NMD efficiencies among cells of the same culture. To investigate the possible causes of the range in NMD efficiency, we sorted cell cultures based on NMD efficiency levels and evaluated cell populations for their concentrations of NMD factors through immunoblotting and RT-qPCR. Results revealed that NMD factor expression levels did not correlate with NMD efficiency, which proposes new questions for the role of NMD factors in NMD and other possible intracellular mechanisms affecting NMD efficiency. We hypothesized that cell cycle may be affecting NMD. To study the possible relationship, groups of cells with varying NMD efficiencies were evaluated through immunoblotting for cell cycle stage. Preliminary results did not indicate a relationship; however, the association must be further evaluated. Conclusively, we determined a range in NMD efficiency among individual cells in homogenous human embryonic kidney cell cultures. We aim to progress this research by determining key factors and mechanisms that may influence NMD efficiency. Implications for understanding the specificities of NMD activity are far-reaching in the medical field, as several severe human diseases, such as facioscapulohumeral muscular dystrophy, are strongly tied to NMD inhibition.