The ribosomal protein Asc1/RACK1 is required for efficient translation of short mRNAs. Saccharomyces cerevisiae Sigma1278b

Translation is a core cellular process carried out by a highly conserved macromolecular machine, the ribosome. There has been remarkable evolutionary adaptation of this machine through the addition of eukaryote-specific ribosomal proteins whose individual effects on ribosome function are largely unknown. Here we show that eukaryote-specific Asc1/RACK1 is required for efficient translation of mRNAs with short open reading frames that show greater than average translational efficiency in diverse eukaryotes. ASC1 mutants in S. cerevisiae display compromised translation of specific functional groups, including cytoplasmic and mitochondrial ribosomal proteins, and display cellular phenotypes consistent with their gene-specific translation defects. Asc1-sensitive mRNAs are preferentially associated with the translational ‘closed loop’ complex comprised of eIF4E, eIF4G and Pab1, and depletion of eIF4G mimics the translational defects of ASC1 mutants. Together our results reveal a role for Asc1/RACK1 in a length-dependent initiation mechanism optimized for efficient translation of genes with important housekeeping functions. Overall design: Ribosome footprint profiling and matched RNA-Seq was performed on S. cerevisiae mutants with altered Asc1 function or expression. Data from additional ribosomal protein mutants, rpl23b∆, rpp1a∆, rps0b∆, and rps16b∆, was collected for comparison. Data was also collected from an ASC1 protein null mutant, asc1-M1X, in a stress condition (growth in glycerol-containing media). In each case, data was collected from two biological replicates. Supplementary processed data files linked below: abbreviations: FP= ribosome footprint profiling total= total RNA-Seq TE= translation efficiency measurements glycerol= yeast grown in rich media containing glycerol instead of glucose rz= libraries made using Ribo-Zero rRNA subtraction rather than polyA selection reprep= library made from the same biological replicate starting material, as noted, in order to obtain higher read coverage. For downstream analyses, the original and reprep libraries were pooled. *counts.txt: Counts per feature for each library. Unique-mapping reads only included. *scaledcounts.txt Counts scaled (per experiment, including replicates) using the DESeq R package. *rpkms.txt: Reads per kilobase per million mapped reads for each feature. *foldchanges.csv: log2 fold changes for all mutants (mutant vs. wild type, average values derived from two biological replicates). *filtered_foldchanges.csv: log2 fold changes of all mutants (mutant vs. wild type, average values derived from two biological replicates), filtered such that each comparison (i.e. mutant vs. WT total RNA-Seq) has a minimum of 128 reads. Features too lowly expressed to meet this cutoff were removed from the relevant comparison.

翻译是一类由高度保守的大分子机器——核糖体（ribosome）所介导的核心细胞过程。该机器通过真核生物特异性核糖体蛋白的添加发生了显著的进化适配，而这些蛋白对核糖体功能的个体效应在很大程度上仍未明确。本研究证实，真核生物特异性蛋白Asc1/RACK1是高效翻译短开放阅读框（open reading frame，ORF）mRNA所必需的；这类mRNA在多种真核生物中展现出高于平均水平的翻译效率。酿酒酵母（S. cerevisiae）中的ASC1突变体表现出特定功能类群的翻译能力受损，包括细胞质与线粒体核糖体蛋白，并呈现出与其基因特异性翻译缺陷相符的细胞表型。对Asc1敏感的mRNA优先与由eIF4E、eIF4G和Pab1组成的翻译‘闭环’复合物结合，而敲除eIF4G会模拟ASC1突变体的翻译缺陷。综上，本研究结果揭示了Asc1/RACK1在依赖于阅读框长度的翻译起始机制中的作用，该机制可优化对具有重要持家功能基因的高效翻译。

实验设计：本研究对Asc1功能或表达发生改变的酿酒酵母（S. cerevisiae）突变体进行了核糖体足迹谱（ribosome footprint profiling）与匹配的RNA测序（RNA-Seq）分析。同时收集了额外的核糖体蛋白突变体（rpl23bΔ、rpp1aΔ、rps0bΔ与rps16bΔ）的数据用于对照。此外还收集了胁迫条件下（在含甘油的培养基中培养）ASC1蛋白敲除突变体asc1-M1X的测序数据。所有样本均设置2个生物学重复。

下文链接的补充处理数据文件的缩写说明如下：
FP：核糖体足迹谱（ribosome footprint profiling）
total：总RNA测序（total RNA-Seq）
TE：翻译效率（translation efficiency）测量值
glycerol：在以甘油而非葡萄糖为碳源的丰富培养基中培养的酵母
rz：采用Ribo-Zero核糖体RNA去除法（而非polyA富集法）构建的文库
reprep：从同一生物学重复起始材料中制备的文库（如前文所述），用于获得更高的测序读段覆盖度。后续分析中，将原始文库与reprep文库进行合并。

各补充数据文件说明如下：
*counts.txt：各文库的每个特征的读段计数，仅包含唯一比对的读段。
*scaledcounts.txt：使用DESeq R包对每个实验（含重复样本）的读段计数进行标准化后的数据。
*rpkms.txt：每个特征的每千碱基每百万比对读段数（reads per kilobase per million mapped reads，RPKM）。
*foldchanges.csv：所有突变体（突变型 vs. 野生型）的log2倍数变化值，数值为2个生物学重复的平均值。
*filtered_foldchanges.csv：所有突变体（突变型 vs. 野生型）的log2倍数变化值，数值为2个生物学重复的平均值；该数据集经过过滤，确保每个比对组（即突变型 vs. 野生型总RNA-Seq）的读段数至少为128。表达量过低无法满足该阈值的特征将被从对应比对分析中移除。