ABSTRACT

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.

摘要

tRNA 表达的差异与大量生物过程有关。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截然不同的作用，但对 tRNA 进行测序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会干扰 RNA-seq 文库制备方法，并限制了高通量测序技术的实用性。在这里，我们通过用去甲基化酶 AlkB 处理并与 tRNA 特异性接头连接，对标准 RNA-seq 方法进行了两种修改，以便对来自四种开花植物的 tRNA 进行测序，一组已被证明具有一些最广泛的转录后 tRNA 修饰率。该协议具有检测全长 tRNA 和序列变体的优势，可用于推断许多转录后修饰。我们使用所得数据生成了几乎所有唯一参考 tRNA 的修改索引拟南芥 (Arabidopsis thaliana)与人类表现出许多古老的保守相似性，但也显示出被子植物 tRNA 修饰的“热点”位置。我们还发现基于 Northern 印迹分析和液滴数字 PCR 的证据表明，即使经过去甲基化处理，tRNA-seq 也可能产生高度偏倚的绝对表达水平估计值，这很可能是由于偏向逆转录造成的。尽管如此，具有修饰数据的全长 tRNA 序列的生成仍然有望用于评估不同治疗、组织或亚细胞组分之间相对 tRNA 表达的差异，并有助于阐明 tRNA 修饰的功能作用。