Observation of MRNA Surveillance in Living Yeast by Ribosome Profiling

Abstract

The synthesis of proteins is a major step in gene expression and therefore represents a regulatory point for modulating cellular output. During elongation, ribosomes are thought to pause along the mRNA transcript upon colliding with various obstacles or translating specific sequence motifs. In addition, ribosomes halt if they arrive at the 3’ end of a transcript after failing to properly terminate or if the message is endonucleolytically cleaved. Genetic studies have suggested that the protein Dom34 and a GTPase binding partner, Hbs1, target mRNAs associated with paused ribosomes for degradation (Doma and Parker 2006, Nature 440:461-4). While biochemical data support this idea by showing that Dom34 directly binds and dissociates ribosomes (Shoemaker et al. 2010, Science 330:369-72), the natural cellular targets of Dom34 remain unknown. To identify functions of Dom34 in vivo, knockout and wild type strains of S. cerevisiae were subjected to a high-throughput footprinting analysis (ribosome profiling) that reveals the places where ribosomes are bound throughout the transcriptome. These data reveal that ribosome occupancy on known pauses is not generally enhanced in the strain lacking Dom34. Instead, the targets of Dom34 include a small number of specialized targets in open reading frames. In addition, ribosomes are enriched at the 3’ end of many transcripts in the Dom34 deletion strain and generally covere <10 bp of polyadenine at the 3’ end of the protected fragment. These data imply that Dom34 dissociates ribosomes that bypass the stop codon and thus serves as a quality control factor required for recycling ribosomes that fail to terminate by the conventional pathway (mediated by eRF1, eRF3, and Rli1). These results support a general role for Dom34 in ribosome recycling in addition to a more specialized role on particular gene products.