Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTR
- University of Massachusetts Medical School, United States
- University of California, Berkeley, United States
Abstract
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.
Data availability
Deep sequencing data are available at GEO, accession #GSE197651
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Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTRNCBI Gene Expression Omnibus, GSE197651.
Article and author information
Author details
Funding
National Institutes of Health (R01HD099816)
- Hans Tobias Gustafsson
- Lucas Ferguson
- Carolina Galan
- Tianxiong Yu
- Heather Upton
- Ebru Kaymak
- Zhiping Weng
- Kathleen Collins
- Oliver J Rando
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Animal husbandry and experimentation was reviewed, approved, and monitored under the University of Massachusetts Medical School Institutional Animal Care and Use Committee (Protocol ID: A-1833-18).
Copyright
© 2025, Gustafsson et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTR
eLife 14:e77616.
https://doi.org/10.7554/eLife.77616
Further reading
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