RNA-Seq and Ribosome Profiling in Saccharomyces cerevisiae

High-throughput sequencing provides a method to globally analyze the transcriptome of an organism. We have employed several sequencing approaches to investigate the transcriptome of Saccharomyces cerevisiae. Strand-specific, random-primed RNA-Seq of whole-cell (rRNA-depleted) RNA from wild-type yeast was performed to measure steady-state gene expression and investigate both annotated and unannotated RNAs. To interrogate the translational state of all RNAs, RNA-Seq of RNAs which sediment with polyribosomes in a sucrose gradient (i.e. Polysome-Seq) was performed in parallel. This analysis was coupled with ribosome profiling, to identify the binding sites of ribosomes on distinct classes of RNAs at nucleotide-level resolution. Finally, RNA-seq performed on a mutant yeast strain deficient in the translation-dependent nonsense-mediated RNA decay pathway (NMD; upf1?) provided a functional readout for the translation of RNAs. Additionally, global identification of targets of this quality control pathway may allow the elucidation of NMD-targetting characteristics. This study will provide global information about the translational status of distinct classes of yeast RNAs and expand the understanding of how RNAs are targeted for NMD quality control.

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