Lso2 is a conserved ribosome-bound protein required for translational recovery in yeast. 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 (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. 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组。
本研究通过定量质谱（quantitative mass spectrometry）鉴定出晚期注释短开放阅读框2（Late-annotated short open reading frame 2, Lso2）为一类在真核生物中广泛保守的核糖体相关蛋白。对Lso2及其人类同源物CCDC124开展全基因组交联免疫沉淀实验，在A位点（A site）附近覆盖GTP酶激活中心（GTPase activating center）的区域富集到25S核糖体RNA（25S rRNA）。与该定位一致，Lso2同时可与绝大多数转运RNA（tRNA）发生交联。
对缺失LSO2的酵母（lso2Δ）进行核糖体谱分析（ribosome profiling）发现，酵母从静止期恢复过程中存在全局性翻译缺陷，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聚集、在终止密码子处耗竭，且lso2Δ菌株中核糖体驻留呈现密码子特异性改变。
上述缺陷以及Lso2/CCDC124特异性核糖体结合活性的保守性，提示二者在翻译过程与生理活动中发挥广泛且关键的功能。
总体实验设计：
增强型交联免疫沉淀（enhanced crosslinking and immunoprecipitation, IP）联合深度测序：包含5组Lso2与CCDC124的IP样本，以及6组分子量匹配的输入样本与未标记IP对照样本。
核糖体谱分析：包含7组野生型（wild-type）样本与7组lso2缺失突变体样本。