Transcriptome-wide analysis of roles for tRNA modifications in translational regulation. Saccharomyces cerevisiae

Covalent nucleotide modifications in noncoding RNAs such as tRNAs affect a plethora of biological processes, with new functions continuing to be discovered for even well-known tRNA modifications. To systematically compare the functions of a large set of ncRNA modifications in gene regulation, we carried out ribosome profiling and RNA-Seq in budding yeast for 57 nonessential genes involved in tRNA modification. Overall design: Yeast strains were treated with cycloheximide and proceeded to ribosome profiling. For ribosome-protected footprints (RPF), 80S monosome fractions were isolated after RNase I treatment, and 27-34 nt RPF were isolated by denaturing PAGE. For RNA-Seq, total RNA was depleted of rRNA using Ribo-Zero, followed by zinc-based fragmentation. RNA fragments and RPF are constructed into Illumina deep-sequencing libraries by RNA 3’ adaptor ligation and cDNA circularization. Barcoded libraries were sequenced on an Illumina NextSeq 500. Raw fastq reads were de-multiplexed and removed of adaptor sequence. RPF reads were mapped to S. cerevisiae rDNA and the mapping reads were discarded. The remaining RPF reads and RNA-seq reads were mapped to sacCer3 genome. Uniquely mapping reads in length of 27-34 nt (RPF) or 27 nt (RNA-seq) were quantified as RPKM.

转运RNA（tRNA）这类非编码RNA（ncRNA）所发生的共价核苷酸修饰，可调控众多生物学过程；即便针对已被广泛研究的tRNA修饰，其全新生物学功能仍在持续被发掘。为系统性对比一大批非编码RNA修饰在基因调控中的功能，我们针对酿酒酵母中57个参与tRNA修饰的非必需基因，开展了核糖体谱（ribosome profiling）与RNA测序（RNA-Seq）实验。整体实验设计如下：先用放线菌酮处理酵母菌株，随后开展核糖体谱实验。针对核糖体保护片段（RPF）：经核糖核酸酶I（RNase I）处理后分离得到80S单核糖体组分，再通过变性聚丙烯酰胺凝胶电泳（PAGE）筛选出长度为27~34 nt的RPF。针对RNA-Seq：先使用Ribo-Zero试剂盒去除总RNA中的核糖体RNA（rRNA），再采用锌离子介导的方法进行RNA片段化。通过RNA 3’端接头连接与互补DNA（cDNA）环化，将RNA片段与RPF构建为Illumina深度测序文库。将带条形码的测序文库置于Illumina NextSeq 500测序平台上进行测序。对原始fastq格式测序reads进行去多重条形码拆分，并移除接头序列。将RPF reads比对至酿酒酵母核糖体DNA（rDNA）序列，丢弃成功比对上的reads。将剩余的RPF reads与RNA-seq reads比对至sacCer3参考基因组。对长度为27~34 nt的唯一比对RPF，以及27 nt的唯一比对RNA-seq reads，以每百万reads每千碱基片段数（RPKM）进行定量。