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 CCDC124 recovered 25S rRNA in a region near the A site that overlaps the GTPase activating center. Consistent with this location, Lso2 also crosslinked to most tRNAs. Ribosome profiling of yeast lacking LSO2 (lso2?) 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 lso2?. These defects, and the conservation of the specific ribosome-binding activity of Lso2/CCDC124, indicate broadly important functions in translation and physiology. Overall design: Enhanced crosslinking and immunoprecipitation (IP) followed by deep sequencing: 5 IP samples of Lso2 and CCDC124, 6 controls of size-matched input and untagged IP. Ribosome footprint profiling: 7 samples of wild-type and 7 samples of lso2 null mutants.核糖体结合蛋白广泛参与蛋白质合成、基因调控与细胞稳态，但目前仍未完整解析所有具有功能的核糖体结合蛋白完整组分。本研究借助定量质谱技术，将晚期注释短开放阅读框2（Late-annotated short open reading frame 2, Lso2）鉴定为一类在真核生物中广泛保守的核糖体相关蛋白。全基因组交联免疫沉淀实验针对Lso2及其人类同源物CCDC124开展分析，在靠近A位点、覆盖GTP酶激活中心的区域富集到25S核糖体RNA（rRNA）。与该结合位置一致，Lso2同时可与绝大多数转运RNA（transfer RNA, tRNA）发生交联。对缺失LSO2基因的酿酒酵母（lso2缺失突变体）进行核糖体足迹谱（ribosome profiling）分析发现，酵母在从静止期恢复生长的过程中出现全局翻译缺陷，多数基因的翻译水平下调幅度超过4倍。核糖体在起始密码子处发生聚集，于终止密码子处出现丰度耗竭，且lso2缺失突变体的核糖体占据度呈现密码子特异性改变。上述缺陷，以及Lso2/CCDC124所具有的保守核糖体结合活性，均提示二者在翻译过程与生理功能中发挥广泛且关键的作用。实验总体设计：增强型交联免疫沉淀（IP）联合深度测序：包含5组针对Lso2与CCDC124的免疫沉淀样本，以及6组尺寸匹配的输入对照与未标签免疫沉淀对照样本。核糖体足迹谱测序：包含7组野生型样本与7组lso2基因缺失突变体样本。