Combining tRNA sequencing methods to characterize plant tRNA expression and post-transcriptional modification
Combining tRNA sequencing methods to characterize plant tRNA expression and post-transcriptional modification
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DOI：
10.1080/15476286.2020.1792089
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发表时间：
2021-01-02
期刊：
影响因子：
4.1
通讯作者：
Sloan, Daniel B.
中科院分区：
文献类型：
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作者：
Warren, Jessica M.;Salinas-Giege, Thalia;Sloan, Daniel B.
tRNA表达的差异与许多生物学过程有关。越来越多的证据表明，tRNA基因可以根据表达和转录后修饰发挥显着不同的作用，但测序tRNA以测量丰度和检测修饰仍然具有挑战性。它们的二级结构和广泛的转录后修饰干扰RNA-seq文库制备方法，并限制了高通量测序技术的应用。在这里，我们通过使用去甲基化酶AlkB和连接tRNA特异性适配器，将两种修饰结合到标准RNA-seq方法中，以便对四种开花植物的tRNA进行测序，这四种开花植物已被证明具有一些最广泛的转录后tRNA修饰率。该方案具有检测全长trna和序列变异的优势，可用于推断许多转录后修饰。我们利用结果数据生成了拟南芥中几乎所有独特参考trna的修饰指数，这些参考trna与人类具有许多古老的保守相似性，但也有一些位置似乎是angioplant trna修饰的“热点”。我们还发现基于northern blot分析和液滴数字PCR的证据表明，即使在去甲基化处理后，tRNA-seq也可能产生高度偏倚的绝对表达水平估计，这很可能是由于偏倚的逆转录。尽管如此，生成具有修饰数据的全长tRNA序列仍然有望评估不同治疗、组织或亚细胞组分中相对tRNA表达的差异，并有助于阐明tRNA修饰的功能作用。
Differences in tRNA expression have been implicated in a remarkable number of biological processes. There is growing evidence that tRNA genes can play dramatically different roles depending on both expression and post-transcriptional modification, yet sequencing tRNAs to measure abundance and detect modifications remains challenging. Their secondary structure and extensive post-transcriptional modifications interfere with RNA-seq library preparation methods and have limited the utility of high-throughput sequencing technologies. Here, we combine two modifications to standard RNA-seq methods by treating with the demethylating enzyme AlkB and ligating with tRNA-specific adapters in order to sequence tRNAs from four species of flowering plants, a group that has been shown to have some of the most extensive rates of post-transcriptional tRNA modifications. This protocol has the advantage of detecting full-length tRNAs and sequence variants that can be used to infer many post-transcriptional modifications. We used the resulting data to produce a modification index of almost all unique reference tRNAs in Arabidopsis thaliana, which exhibited many anciently conserved similarities with humans but also positions that appear to be 'hot spots' for modifications in angiosperm tRNAs. We also found evidence based on northern blot analysis and droplet digital PCR that, even after demethylation treatment, tRNA-seq can produce highly biased estimates of absolute expression levels most likely due to biased reverse transcription. Nevertheless, the generation of full-length tRNA sequences with modification data is still promising for assessing differences in relative tRNA expression across treatments, tissues or subcellular fractions and help elucidate the functional roles of tRNA modifications.