Development of High-throughput 5Pseq and detection of ribosome stalls at termination level in Saccharomyces cerevisiae

We developed a high throughput 5'P sequencing approach (HT-5PSeq) to investigate 5'P mRNA degradome in relation with translation process. This approach is easy and scalable; and uses an affordable rRNA depletion based on duplex-specific nuclease treatment. We use it to investigate in vivo ribosome stalls in S. cerevisiae and S. pombe at single nucleotide resolution. We investigate the 5'P degradation profiles associated to ribosome pausing, its regulation in stress conditions and the relative poly(A) length of mRNA degradation intermediates. Overall design: We performed 2 biologically independent HT-5PSeq experiments. For S.cerevisiae, we performed HT-5Pseq with DSN treatment with probes targeting ribosomal rRNA (BY-DSN) and none DSN treatment (BY-NonDSN) , DSN treatment without probes (BY-NP) as control. We also performed 5Pseq (PMID: 26046441) in S.cerevisiae as positive control. For S.pombe, we performed HT-5Pseq with DSN treatment with probes (orginally designed for S.cerevisiae ribosomal rRNA) (Pombe-DSN) and none DSN treatment (Pombe-NonDSN) as control.To inhibit translation process, we used cycloheximide (CHX) as a translation inhibitor on S.cerevisiae (BY-CHX) and S.pombe (Pombe-CHX). In addition, we performed HT-5Pseq under different growth conditions. For early exponential phase, strains were grown to a final OD600 of 0.3 (BY-lowOD). To reach stationary stage, strains were grown during 60h in YPD (BY-Stationary). We performed paired-end sequencing for read1 (61bp) and read2 (15bp) in some samples (see below). We splited libraries with polyA and non-polyA based on the length of As in read2. For other samples, we performed single end sequencing.

本研究开发了一种高通量5'端磷酸化测序方法（HT-5PSeq），用于探究与翻译过程相关的5'端磷酸化mRNA降解组。该方法操作简便且可规模化应用，基于双链特异性核酸酶（duplex-specific nuclease, DSN）处理实现成本可控的核糖体RNA（ribosomal RNA, rRNA）去除。我们利用该方法以单核苷酸分辨率，探究酿酒酵母（Saccharomyces cerevisiae, S. cerevisiae）和粟酒裂殖酵母（Schizosaccharomyces pombe, S. pombe）体内的核糖体停滞现象，并分析与核糖体暂停相关的5'端磷酸化降解特征、其在应激条件下的调控机制，以及mRNA降解中间体的相对poly(A)尾长度。

实验整体设计：本研究共实施2组生物学独立的HT-5PSeq实验。针对酿酒酵母，我们设置了以下实验组与对照组：采用靶向核糖体rRNA的探针结合DSN处理的HT-5PSeq实验（BY-DSN）、无DSN处理的HT-5PSeq实验（BY-NonDSN），以及仅进行DSN处理、未添加探针的HT-5PSeq实验（BY-NP）作为对照；同时以酿酒酵母为材料开展5'P测序（5Pseq，PMID: 26046441）作为阳性对照。针对粟酒裂殖酵母，我们采用原本为酿酒酵母核糖体rRNA设计的探针结合DSN处理开展HT-5PSeq实验（Pombe-DSN），并设置无DSN处理的HT-5PSeq实验（Pombe-NonDSN）作为对照。为抑制翻译过程，我们分别向酿酒酵母（BY-CHX）和粟酒裂殖酵母（Pombe-CHX）培养基中添加翻译抑制剂环己酰亚胺（cycloheximide, CHX）。此外，本研究在不同生长条件下开展HT-5PSeq实验：对于早期指数生长期菌株，将其培养至最终OD₆₀₀为0.3（BY-lowOD）；为获得稳定期菌株，将菌株在YPD培养基中培养60小时（BY-Stationary）。部分样本采用双端测序策略，对读段1（read1，61bp）和读段2（read2，15bp）进行测序（详见下文）。我们根据读段2中的腺嘌呤（A）序列长度，将文库分为含poly(A)尾与不含poly(A)尾两类。其余样本则采用单端测序策略。