Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTR

  1. Hans Tobias Gustafsson
  2. Lucas Ferguson
  3. Carolina Galan
  4. Tianxiong Yu
  5. Heather Upton
  6. Ebru Kaymak
  7. Zhiping Weng
  8. Kathleen Collins
  9. Oliver J Rando  Is a corresponding author
  1. University of Massachusetts Medical School, United States
  2. 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

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Hans Tobias Gustafsson

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  2. Lucas Ferguson

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    Lucas Ferguson, is an inventor on published patent applications filed by University of California describing OTTR technology, all of which are also described in peer-reviewed journal publications. Has equity in the company that licensed the OTTR technology (Karnateq)..
  3. Carolina Galan

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  4. Tianxiong Yu

    Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  5. Heather Upton

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  6. Ebru Kaymak

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  7. Zhiping Weng

    Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  8. Kathleen Collins

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    Kathleen Collins, Is an inventor on published patent applications filed by University of California describing OTTR technology, all of which are also described in peer-reviewed journal publications. Has equity in the company that licensed the OTTR technology (Karnateq)..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3172-7088
  9. Oliver J Rando

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    For correspondence
    Oliver.Rando@umassmed.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1516-9397

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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  1. Hans Tobias Gustafsson
  2. Lucas Ferguson
  3. Carolina Galan
  4. Tianxiong Yu
  5. Heather Upton
  6. Ebru Kaymak
  7. Zhiping Weng
  8. Kathleen Collins
  9. Oliver J Rando
(2025)
Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTR
eLife 14:e77616.
https://doi.org/10.7554/eLife.77616

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https://doi.org/10.7554/eLife.77616