UC Berkeley

Regulation of gene expression is critical in maintaining cellular identity throughout an organism’s development or ensuring a single celled organisms’ survival in response to stimuli. Gene expression can be modified through many levels of regulation including control over transcription, translation, and protein stability. In budding yeast, nutrient availability can trigger complex gene regulation programs to influence cellular growth or induce differentiation to optimize survival. Under states of abundant nutrients, cells will divide rapidly through the process of mitosis. In response to low nutrients, cells undergo the developmental program of meiosis. Meiosis is a cell division process that produces specialized haploid gametes from diploid cells and in yeast, produces gametes in the form of spores. Intricate gene expression programs coordinate the massive cellular reprogramming that takes place throughout this differentiation. Work from the Brar and Ünal labs has previously elucidated a non-conventional type of gene regulation used prevalently in meiosis and in response to other stimuli in yeast. This regulation was surprising because for genes regulated in this manner, long undecoded transcript isoforms (LUTIs) are transcribed in place of canonical transcripts, which leads to an anticorrelation between mRNA levels and protein abundance. Because of our knowledge of this non-canonical regulation, I was able to identify a potent and overlooked off-target effect occurring in selection marked yeast mutants, resulting from LUTI-based regulation. In chapter 1 we dissect the mechanism by which this effect occurs and generate improved cassettes that do not exhibit this off-target effect for use in future studies. In chapter 2, I offer a guide of best practices to use the ribosome profiling method to study gene regulation during meiosis in budding yeast. Finally, in chapter 3, I carefully dissect the specific functions and characteristics of two RNA helicases that are expressed in an anticorrelated manner using genome edited mutants and ribosome profiling. In summary, we find that the careful dissection of molecular questions relies on integrated genome editing precision, and carefully controlled experiments and interpretations.