Analysis of Ribosome Protected Fragments of mRNAs from yeast strains either expressing Rio1p or depleted of Rio1p. Saccharomyces cerevisiae

mRNAs bound by ribosomes from yeast cells were analysed in order to determine the exact position of ribosomes in the presence or absence of Rio1p. Beside total Ribosome Protected Fragments (RPFs), RPFs from mRNAs protected by immature pre-40S pre-ribosomes was also analysed. The analysis showed that immature 40S ribosomes can carry out translation and their premature entry into translation is hindered by Rio1p. Overall design: mRNAs fragments protected by ribosomes were subjected to NGS Ribosome Profiling experiment. Samples included (1) total ribosome-associated mRNAs from yeast cells expressing Rio1p, (2) total ribosome-associated mRNAs from yeast cells depleted of Rio1p and (3) immuno-selected mRNAs associated with immature pre-40S pre-ribosomal particles from yeast-cells depleted of Rio1p. Ribosome-protected mRNA fragments were used as templates for cDNA libraries. The resulting cDNAs were sequenced and the sequences were annealed to the yeast genome. The position of the reads were determined in relation to either the translation start site or the stop codon of the corresponding coding DNA sequences.

本研究对酵母细胞中核糖体结合的mRNA开展分析，以明确存在或缺失Rio1p时核糖体的精确定位。除总核糖体保护片段（Ribosome Protected Fragments, RPFs）外，本研究同时分析了未成熟前40S pre-核糖体（pre-40S pre-ribosomes）保护的mRNA来源的RPFs。分析结果表明，未成熟40S核糖体可执行翻译过程，而Rio1p会阻碍其过早进入翻译程序。

整体实验设计：将核糖体保护的mRNA片段进行下一代测序（Next Generation Sequencing, NGS）核糖体谱实验。实验样本包含三类：(1) 表达Rio1p的酵母细胞内总核糖体结合mRNA；(2) Rio1p耗尽的酵母细胞内总核糖体结合mRNA；(3) 经免疫筛选的、来自Rio1p耗尽的酵母细胞的未成熟前40S pre-核糖体颗粒结合的mRNA。以核糖体保护的mRNA片段为模板构建cDNA文库，对所得cDNA进行测序，并将测序序列比对至酵母基因组。根据对应编码DNA序列的翻译起始位点或终止密码子，确定测序读段的基因组定位。