Quantitative Comparisons of Translation Activity by Ribosome Profiling with Internal Standards.

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.

Lso2 is a conserved ribosome-bound protein required for translational recovery in yeast.

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.