RESUMO
Ribosome-binding proteins function broadly in protein synthesis, gene regulation, and cellular homeostasis, but the complete complement of functional ribosome-bound proteins remains unknown. Using quantitative mass spectrometry, we identified late-annotated short open reading frame 2 (Lso2) as a ribosome-associated protein that is broadly conserved in eukaryotes. Genome-wide crosslinking and immunoprecipitation of Lso2 and its human ortholog coiled-coil domain containing 124 (CCDC124) recovered 25S ribosomal RNA in a region near the A site that overlaps the GTPase activation center. Consistent with this location, Lso2 also crosslinked to most tRNAs. Ribosome profiling of yeast lacking LSO2 (lso2Δ) revealed global translation defects during recovery from stationary phase with translation of most genes reduced more than 4-fold. Ribosomes accumulated at start codons, were depleted from stop codons, and showed codon-specific changes in occupancy in lso2Δ. These defects, and the conservation of the specific ribosome-binding activity of Lso2/CCDC124, indicate broadly important functions in translation and physiology.
Assuntos
Sequência Conservada , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Códon de Iniciação/genética , Regulação Fúngica da Expressão Gênica , Células HeLa , Humanos , Elongação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Ribosome profiling is a genome-wide approach to map the positions of ribosomes on messenger RNAs. The abundance of ribosome-protected fragments can be used within condition to compare relative translation activities between different transcripts and between distinct conditions for the same transcript. A unified and routine method is currently lacking, however, to normalize between conditions for differences in global translation levels. Here we describe experimental and computational methods to use an orthogonal species spike-in, or internal standard, to enable absolute comparisons of translation activity between conditions. This simple modification of standard ribosome profiling provides a robust approach for accurately interpreting the effects of diverse genetic, chemical, and environmental perturbations of translation.