Gustafsson Hans Tobias, Ferguson Lucas, Galan Carolina, Yu Tianxiong, Upton Heather, Kaymak Ebru, Weng Zhiping, Collins Kathleen, Rando Oliver J
Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, United States.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.
Elife. 2025 Apr 25;14:e77616. doi: 10.7554/eLife.77616.
Among the major classes of RNAs in the cell, tRNAs remain the most difficult to characterize via deep sequencing approaches, as tRNA structure and nucleotide modifications can each interfere with cDNA synthesis by commonly used reverse transcriptases (RTs). Here, we benchmark a recently developed RNA cloning protocol, termed Ordered Two-Template Relay (OTTR), to characterize intact tRNAs and tRNA fragments in budding yeast and in mouse tissues. We show that OTTR successfully captures both full-length tRNAs and tRNA fragments in budding yeast and in mouse reproductive tissues without any prior enzymatic treatment, and that tRNA cloning efficiency can be further enhanced via AlkB-mediated demethylation of modified nucleotides. As with other recent tRNA cloning protocols, we find that a subset of nucleotide modifications leave misincorporation signatures in OTTR datasets, enabling their detection without any additional protocol steps. Focusing on tRNA cleavage products, we compare OTTR with several standard small RNA-Seq protocols, finding that OTTR provides the most accurate picture of tRNA fragment levels by comparison to 'ground truth' Northern blots. Applying this protocol to mature mouse spermatozoa, our data dramatically alter our understanding of the small RNA cargo of mature mammalian sperm, revealing a far more complex population of tRNA fragments - including both 5' and 3' tRNA halves derived from the majority of tRNAs - than previously appreciated. Taken together, our data confirm the superior performance of OTTR to commercial protocols in analysis of tRNA fragments, and force a reappraisal of potential epigenetic functions of the sperm small RNA payload.
在细胞中的主要RNA类别中，tRNA仍然是通过深度测序方法最难表征的，因为tRNA的结构和核苷酸修饰都会干扰常用逆转录酶（RT）进行的cDNA合成。在这里，我们对一种最近开发的RNA克隆方案——有序双模板接力（OTTR）进行了基准测试，以表征出芽酵母和小鼠组织中的完整tRNA和tRNA片段。我们表明，OTTR无需任何预先的酶处理就能成功捕获出芽酵母和小鼠生殖组织中的全长tRNA和tRNA片段，并且通过AlkB介导的修饰核苷酸去甲基化可以进一步提高tRNA克隆效率。与其他最近的tRNA克隆方案一样，我们发现一部分核苷酸修饰会在OTTR数据集中留下错掺入特征，无需任何额外的方案步骤就能检测到它们。聚焦于tRNA裂解产物，我们将OTTR与几种标准的小RNA测序方案进行了比较，发现与“真实情况”的Northern印迹相比，OTTR能提供最准确的tRNA片段水平情况。将该方案应用于成熟的小鼠精子，我们的数据极大地改变了我们对成熟哺乳动物精子中小RNA含量的理解，揭示了比以前认识到的更为复杂的tRNA片段群体——包括来自大多数tRNA的5'和3'tRNA半体。总之，我们的数据证实了OTTR在分析tRNA片段方面优于商业方案，并促使人们重新评估精子小RNA有效载荷的潜在表观遗传功能。
