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Eser, Philipp (2016): Quantitative genome-wide studies of RNA metabolism in yeast. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



Gene expression and its regulation are fundamental processes in every living cell and organism. RNA molecules hereby play a central role by translating the genetic information into proteins, by regulating gene activity and by forming structural components. The kinetics of RNA metabolism differ widely between genes and conditions and play an important role for cellular processes, but how this is achieved remains poorly understood. Here, we used a novel experimental protocol that allows profiling of newly transcribed RNAs in conjunction with an advanced computational modeling pipeline to explore the kinetics of RNA metabolism and the underlying genetic determinants.In the first study, we investigated cell cycle regulated gene expression and the contributions of synthesis and degradation to mRNA levels in S.cerevisiae. During the cell cycle, the levels of hundreds of mRNAs change in a periodic manner, but how this is carried out by alterations in the rates of mRNA synthesis and degradation has not been studied systematically. We were able to derive mRNA synthesis and degradation rates every 5 minutes during the cell cycle, and thus provide for the first time a high-resolution time series of RNA metabolism during the cell cycle. A novel statistical model identified 479 genes that show periodic changes in mRNA synthesis and generally also periodic changes in their mRNA degradation rates. Peaks of mRNA degradation follow peaks of mRNA synthesis, resulting in sharp and high peaks of mRNA levels at defined times during the cell cycle. Whereas the timing of mRNA synthesis is set by upstream DNA motifs and their associated transcription factors (TFs), the synthesis rate of a periodically expressed gene is apparently set by its core promoter. In the second study, we developed metabolic labeling with RNA-Seq (4tU-Seq) and novel computational methods to gain further insights into the kinetics of RNA metabolism and its regulation. To decrypt the regulatory code of the genome, sequence elements must be defined that determine RNA turnover and thus gene expression. Here we attempt such decryption in an eukaryotic model organism, the fission yeast S. pombe. We first derived an improved genome annotation that redefines borders of 36% of expressed mRNAs and adds 487 non-coding RNAs (ncRNAs). We then combined RNA labeling in-vivo with mathematical modeling to obtain rates of RNA synthesis and degradation for 5,484 expressed RNAs and splicing rates for 4,958 introns. We identified functional sequence elements in DNA and RNA that control RNA metabolic rates, and quantified the contributions of individual nucleotides to RNA synthesis, splicing, and degradation. Our approach reveals distinct kinetics of mRNA and ncRNA metabolism, separates antisense regulation by transcription interference from RNA interference, and provides a general tool for studying the regulatory code of genomes.