Combining tRNA sequencing methods to characterize plant tRNA expression and post-transcriptional modification

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.

转运RNA（transfer RNA，tRNA）的表达差异与大量生物学过程密切相关。越来越多的研究表明，tRNA基因的功能会因其表达水平和转录后修饰状态的不同而发生显著改变，但通过测序手段定量tRNA丰度并检测其修饰位点仍存在诸多挑战。其复杂的二级结构以及广泛存在的转录后修饰会干扰RNA测序（RNA-seq）文库构建流程，极大限制了高通量测序技术在tRNA研究中的应用。本研究对标准RNA-seq方法进行两项改良：使用去甲基化酶AlkB进行处理，并连接tRNA特异性接头，以此对四种显花植物的tRNA进行测序——该类群被证实存在极为广泛的tRNA转录后修饰现象。该实验方案的优势在于可获取全长tRNA序列并鉴定序列变异，进而推断出多数转录后修饰位点。我们利用所得数据为拟南芥（Arabidopsis thaliana）中几乎所有唯一参考tRNA构建了修饰指数，该指数不仅展现出与人类诸多古老保守的相似特征，同时也存在被子植物tRNA修饰的"热点"位点。我们还通过Northern印迹（Northern blot）分析和微滴式数字PCR（droplet digital PCR）验证发现：即便经过去甲基化处理，tRNA测序仍会对绝对表达水平产生显著偏倚的估计，这一现象极有可能源于反转录过程的偏好性。尽管如此，结合修饰数据获取全长tRNA序列的方法，仍在评估不同处理、组织或亚细胞组分中tRNA相对表达差异方面具有良好应用前景，同时有助于阐明tRNA修饰的生物学功能。