Poster abstracts
Poster number 72 submitted by Brittany Stawicki
Metabolic labeling of RNA in synchronized S. cerevisiae daughter cells
Brittany N Stawicki (Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH), Eckhard Jankowsky (Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH)
Abstract:
RNA metabolism markedly changes during the cell cycle. A large percentage of mRNAs undergo translational repression during cell division. Equivalent variations in mRNA stability are expected. However, it is currently unknown how exactly translational state and mRNA half lives change with the cell cycle in S. cerevisiae. To address this problem we are devising an approach to measure mRNA half lives and translational state of mRNAs during the cell cycle in S. cerevisiae. Given that mother and daughter cells in S. cerevisiae differ in cell size and cell cycle length, it is also important to separately analyze mother and daughter cells. To isolate synchronized S. cerevisiae daughter cells in a non-disruptive fashion, we attached unsynchronized yeast cells to a coated glass substrate and eluted budding daughter cells in short time intervals. This approach currently yields more than 80% synchronized daughter cells. We then combined this synchronization approach with optimized 4-thiouracil metabolic RNA labeling. We provide evidence that 4-thiouracil labeled RNA enters the translating RNA pool and is effectively translated. However, labeling times of more than 60 min compromise translation. Our combined cell synchronization and metabolic labeling approach is suitable for high-throughput analysis and likely for other S. cerevisiae strains, including those with genetic alterations. The approach sets the stage for quantitative measurements of translational states and mRNA stabilities over the course of the S. cerevisiae cell cycle.
Keywords: Metabolic Labeling, S Cerevisiae