UCLA

Eukaryotic organisms have evolved complex gene regulatory networks to launch coordinated responses to external conditions and stimuli. Under environmental stress, such as nutrient depletion, these responses involve reallocation of cellular resources away from the products of growth and cell cycle stimulating genes and towards the products of stress-responsive genes. This occurs through several mechanisms, including changes in transcription, decreased ribosome biogenesis, altered translation initiation, and changes in mRNA features that modulate translation efficiency and transcript stability. Upon nutrient starvation in the yeast Saccharomyces cerevisiae, diploid cells undergo meiosis leading to sporulation. Previous studies show that the catalytic component of the Swi/Snf chromatin remodeling complex, Snf2, is responsible for shifting gene expression away from intron-rich ribosomal protein genes (RPGs) to enhance splicing of meiotic intron-containing genes (ICGs) during sporulation, and that a similar process occurs during the transition from growth to quiescence known as the diauxic shift in yeast. During both of these complex cell-state transitions, Snf2 protein levels change dramatically while SNF2 mRNA levels remain relatively stable.The aim of this study is to understand the mechanism by which Snf2 protein levels change in response to nutrients. Here I describe alternate transcription start sites (TSS) at the SNF2 gene locus under batch growth conditions, which produce SNF2 transcripts with distinct 5’ leaders affecting downstream translation propensity. Specifically, a long transcript isoform of SNF2 mRNA containing three upstream open reading frames (uORFs), is capable of inhibiting the translation of the downstream protein-coding ORF. I identified the previously unannotated TSS of the long SNF2 isoform and performed RNA analysis in mutant and wild-type cells under various conditions to demonstrate that the transcript isoforms undergo nutrient-responsive transcript isoform switching, which is affected by the transcriptional regulator Ume6. Parallel protein analysis via Western blotting shows that this regulation indeed affects Snf2 expression, and that there is an inverse relationship between expression of the long SNF2 isoform and Snf2 protein levels. In light of the conservation of Snf2-family proteins, investigating SNF2 regulation in response to environmental changes in S. cerevisiae may carry important implications for understanding the regulation of Swi/Snf chromatin remodeling activity in higher eukaryotes.