Tunable protein synthesis by transcript isoforms in human cells (Transcript Isoforms in Polysomes sequencing: TrIP-seq)

Eukaryotic genes generate multiple mRNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell-type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels. Total cytoplasmic and eight polysomal fractions of RNA were purified from HEK 293T cells in biological duplicate. Ribosomal RNA was depleted using Ribo-Zero (Human/Mouse/Rat; Epicenter) and libraries were prepared using the TruSeq RNA v2 kit (RS-122-2001; Illumina) skipping the polyA selection step. Reads are paired-end 75bp and sequencing adapters are GATCGGAAGAGCACACGTCTGAACTCCAGTCAC (read1) and AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT (read2).

真核基因可通过可变转录、可变剪接及多聚腺苷酸化产生多种mRNA转录本亚型（transcript isoforms）。然而，由于不同转录本亚型的翻译过程存在差异，人类转录本多样性与蛋白质生成之间的关联极为复杂。我们对多聚核糖体谱（polysome profile）进行分级分离，并从每个分级组分中重建转录本亚型，将该实验方法命名为多聚核糖体中转录本亚型测序（Transcript Isoforms in Polysomes sequencing, TrIP-seq）。对该数据集的分析揭示了调控每个转录本亚型的核糖体占用率与翻译产出的调控特征。我们提取得到一组5′端与3′端非翻译区（untranslated regions, UTRs），它们可在细胞中对无关基因的蛋白质生成实现100倍以上的调控幅度。部分5′端非翻译区可在不同细胞系间发挥强效的翻译调控作用，而3′端非翻译区则可赋予细胞类型特异性的基因表达特征。这些研究结果揭示了转录本亚型特异性翻译调控的广阔动态范围，鉴定出了人类细胞中调控蛋白质产出的亚型特异性序列，并证实了在关联RNA与蛋白质水平时，必须考虑转录本亚型的多样性。我们从HEK 293T细胞中纯化得到总细胞质RNA与8个多聚核糖体分级组分RNA，实验设置生物学重复两次。我们使用Ribo-Zero（Human/Mouse/Rat; Epicenter）试剂盒去除核糖体RNA，并采用TruSeq RNA v2试剂盒（RS-122-2001; Illumina）构建测序文库，跳过了polyA富集步骤。测序读段为75bp双端读段，测序接头序列分别为：read1接头GATCGGAAGAGCACACGTCTGAACTCCAGTCAC，read2接头AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT。