Article

A transfer-RNA-derived small RNA regulates ribosome biogenesis

  • Nature volume 552, pages 5762 (07 December 2017)
  • doi:10.1038/nature25005
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Abstract

Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3′tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer.

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References

  1. 1.

    & Slicing tRNAs to boost functional ncRNA diversity. RNA Biol. 10, 1798–1806 (2013)

  2. 2.

    & Stressing out over tRNA cleavage. Cell 138, 215–219 (2009)

  3. 3.

    , , & Angiogenin cleaves tRNA and promotes stress-induced translational repression. J. Cell Biol. 185, 35–42 (2009)

  4. 4.

    et al. Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers. Proc. Natl Acad. Sci. USA 112, E3816–E3825 (2015)

  5. 5.

    et al. Sperm tsRNAs contribute to intergenerational inheritance of an acquired metabolic disorder. Science 351, 397–400 (2016)

  6. 6.

    et al. Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals. Science 351, 391–396 (2016)

  7. 7.

    , & Biogenesis and function of transfer RNA-related fragments (tRFs). Trends Biochem. Sci. 41, 679–689 (2016)

  8. 8.

    et al. Stem cell function and stress response are controlled by protein synthesis. Nature 534, 335–340 (2016)

  9. 9.

    , , , & Angiogenin-induced tRNA fragments inhibit translation initiation. Mol. Cell 43, 613–623 (2011)

  10. 10.

    , , & LTR-retrotransposon control by tRNA-derived small RNAs. Cell 170, 61–71 (2017)

  11. 11.

    , & tRNA-derived small RNAs target transposable element transcripts. Nucleic Acids Res. 45, 5142–5152 (2017)

  12. 12.

    et al. Human tRNA-derived small RNAs in the global regulation of RNA silencing. RNA 16, 673–695 (2010)

  13. 13.

    et al. tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma. Proc. Natl Acad. Sci. USA 110, 1404–1409 (2013)

  14. 14.

    , , & Meta-analysis of tRNA derived RNA fragments reveals that they are evolutionarily conserved and associate with AGO proteins to recognize specific RNA targets. BMC Biol. 12, 78 (2014)

  15. 15.

    , & Locked nucleic acid: a potent nucleic acid analog in therapeutics and biotechnology. Oligonucleotides 14, 130–146 (2004)

  16. 16.

    et al. LNA-mediated microRNA silencing in non-human primates. Nature 452, 896–899 (2008)

  17. 17.

    , & Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol. Annu. Rev. 11, 127–152 (2005)

  18. 18.

    et al. ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments. Nat. Methods 12, 879–884 (2015)

  19. 19.

    et al. Efficient and quantitative high-throughput tRNA sequencing. Nat. Methods 12, 835–837 (2015)

  20. 20.

    Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35, 495–516 (2007)

  21. 21.

    & Dissociation of mammalian polyribosomes into subunits by puromycin. Proc. Natl Acad. Sci. USA 68, 390–394 (1971)

  22. 22.

    et al. The role of human ribosomal proteins in the maturation of rRNA and ribosome production. RNA 14, 1918–1929 (2008)

  23. 23.

    & The Myc trilogy: lord of RNA polymerases. Nat. Cell Biol. 7, 215–217 (2005)

  24. 24.

    & Diamond-Blackfan anemia: a ribosomal puzzle. Haematologica 93, 1601–1604 (2008)

  25. 25.

    , & Ribosome biogenesis and control of cell proliferation: p53 is not alone. Cancer Res. 72, 1602–1607 (2012)

  26. 26.

    , & Chromatin isolation by RNA purification (ChIRP). J. Vis. Exp. 2012, 3912 (2012)

  27. 27.

    et al. Regulation of microRNA-mediated gene silencing by microRNA precursors. Nat. Struct. Mol. Biol. 21, 825–832 (2014)

  28. 28.

    , , & Fast and effective prediction of microRNA/target duplexes. RNA 10, 1507–1517 (2004)

  29. 29.

    & Memory efficient folding algorithms for circular RNA secondary structures. Bioinformatics 22, 1172–1176 (2006)

  30. 30.

    Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003)

  31. 31.

    & RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics 11, 129 (2010)

  32. 32.

    et al. Structure prediction: new insights into decrypting long noncoding RNAs. Int. J. Mol. Sci. 17, 132 (2016)

  33. 33.

    et al. RNA duplex map in living cells reveals higher-order transcriptome structure. Cell 165, 1267–1279 (2016)

  34. 34.

    et al. Reduced expression of ribosomal proteins relieves microRNA-mediated repression. Mol. Cell 46, 171–186 (2012)

  35. 35.

    et al. Proliferation, but not growth, blocked by conditional deletion of 40S ribosomal protein S6. Science 288, 2045–2047 (2000)

  36. 36.

    et al. Novel celastrol derivatives inhibit the growth of hepatocellular carcinoma patient-derived xenografts. Oncotarget 5, 5819–5831 (2014)

  37. 37.

    , & Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 6, 1258–1266 (1999)

  38. 38.

    , & High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum. Gene Ther. 10, 1735–1737 (1999)

  39. 39.

    , & TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009)

  40. 40.

    et al. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010)

  41. 41.

    et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protocols 7, 562–578 (2012)

  42. 42.

    , , , & Proteomic analysis of ribosomes: translational control of mRNA populations by glycogen synthase GYS1. J. Mol. Biol. 410, 118–130 (2011)

  43. 43.

    et al. Hematopoietic stem cell quiescence is maintained by compound contributions of the retinoblastoma gene family. Cell Stem Cell 3, 416–428 (2008)

  44. 44.

    , , , & Alternative pre-rRNA processing pathways in human cells and their alteration by cycloheximide inhibition of protein synthesis. Eur. J. Biochem. 212, 211–215 (1993)

  45. 45.

    et al. Mutation of ribosomal protein RPS24 in Diamond-Blackfan anemia results in a ribosome biogenesis disorder. Hum. Mol. Genet. 17, 1253–1263 (2008)

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Acknowledgements

We thank J. Sage for HCC tissues from conditional TKO (Rblox/lox; p130lox/lox; p107−/−) adult mice and liver tissues from p107−/− mice. This work was supported by grants to M.A.K. from the National Institutes of Health (R01AI071068 and R01DK114483). M.A.K. received support from the Stanford Cancer Institute, and S.S. from the CJ Huang Foundation and the TS Kwok Liver Cancer Foundation.

Author information

Author notes

    • Gabriele Fuchs
    • , Shengchun Wang
    • , Yue Zhang
    •  & Biswajoy Roy-Chaudhuri

    Present addresses: The RNA Institute and Department of Biological Sciences, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, USA (G.F.); Medtronic Vascular, 3576 Unocal Place, Santa Rosa, California 95403, USA (S.W.); Stanford Center for Genomics and Personalized Medicine, 3165 Porter Drive, Palo Alto, California 94304, USA (Y.Z.); Impossible Foods Inc., 525 Chesapeake Drive, Redwood City, California 94063, USA (B.R.-C.).

Affiliations

  1. Department of Pediatrics, Stanford University, Stanford, California 94305, USA

    • Hak Kyun Kim
    • , Shengchun Wang
    • , Yue Zhang
    • , Hyesuk Park
    • , Biswajoy Roy-Chaudhuri
    • , Jianpeng Xu
    • , Kirk Chu
    • , Feijie Zhang
    •  & Mark A. Kay
  2. Department of Genetics, Stanford University, Stanford, California 94305, USA

    • Hak Kyun Kim
    • , Shengchun Wang
    • , Yue Zhang
    • , Hyesuk Park
    • , Biswajoy Roy-Chaudhuri
    • , Jianpeng Xu
    • , Kirk Chu
    • , Feijie Zhang
    •  & Mark A. Kay
  3. Department of Microbiology and Immunology, Stanford University, Stanford, California 94305, USA.

    • Gabriele Fuchs
    •  & Peter Sarnow
  4. Asian Liver Center, Department of Surgery, Stanford University School of Medicine, Stanford, California 94305, USA

    • Wei Wei
    • , Mei-Sze Chua
    •  & Samuel So
  5. MOE Key Laboratory of Bioinformatics, Beijing Advanced Innovation Center for Structural Biology, Center for Synthetic and Systems Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China

    • Pan Li
    •  & Qiangfeng Cliff Zhang

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Contributions

H.K.K. contributed to experimental design, interpretation, execution, and manuscript writing and editing. G.F. performed experiments in Fig. 3a, b and Extended Data Fig. 1g, h, and assisted with interpretation, discussion and manuscript editing. S.W. performed experiments in Figs 1c, 2b and Extended Data Figs 1i, 2c. W.W. designed experiments with the PDX model and conducted experiments in Fig. 2c and Extended Data Fig. 2h, i, k. Y.Z. performed computational analyses (RNA-seq and predictions of tsRNA binding sites on pre-45S rRNA) in Extended Data Figs 3d and 5a. H.P. performed multiple experiments including Figs 2a, d, 4a, e, and 5b. B.R.-C. conducted the experiment in Extended Data Fig. 8a. P.L. analysed icSHAPE data in Extended Data Fig. 9d. J.X. performed LeuCAG3′tsRNA target site prediction in Extended Data Figs 8b and 9b. F.Z. performed RNA extraction and mouse experiments. K.C. conducted protein extraction. M.-S.C. designed experiments with the PDX model, interpreted animal data and assisted in manuscript editing. S.S. provided discussion regarding the xenograft model. Q.C.Z. analysed, interpreted and discussed icSHAPE data. P.S. interpreted and discussed experimental results and assisted in manuscript editing. M.A.K. contributed to the experimental design, data interpretation, and manuscript writing and editing.

Competing interests

H.K.K., S.W. and M.A.K. are inventors on relevant patents filed by Stanford University.

Corresponding author

Correspondence to Mark A. Kay.

Reviewer Information Nature thanks N. Polacek and L. Zender for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Life Sciences Reporting Summary

  2. 2.

    Supplementary Figure 1

    This file contains gel source data.

  3. 3.

    Supplementary Table 1

    This file contains the DNA sequences of the CUG (original) and CUC/CUU (modified) Renilla genes from the psiCHECK-2 plasmid from Fig. 1c.Red colored nucleotides show the LeuCUG codon and blue colored nucleotides are LeuCUU or LeuCUC codons. There are thirteen LeuCUG, four LeuCUU, and five LeuCUC codons in original Renilla gene. Thirteen LeuCUG codons were replaced by CUU or CUC codons in the modified Renilla gene.

  4. 4.

    Supplementary Table 2

    This file contains samples that were sequenced in Extended Data Fig. 3d. 50bp paired-end reads were generated on an Illumina HiSeq 2000 machine yielding a total of 10 to 40 million paired-end reads. Sequences were mapped to the human hg19 genome.

  5. 5.

    Supplementary Table 3

    This file contains quantification of each ribosomal RNA in Fig. 3d. Each pre-rRNA was normalized by each mature 28S rRNA. Normalized pre-rRNA from Anti-Leu3′ts LNA was again normalized to that of control (con). Normalized pre-rRNA from siRPS6, 10, 13, and 29 were normalized to the siRNA control (sicontrol).

  6. 6.

    Supplementary Table 4

    This file contains the list of antisense Locked nucleic acid (LNA), synthetic RNA, and northern probe oligonucleotides. LNA bases are upper-case letters and DNA bases are lower –case letters.

  7. 7.

    Supplementary Table 5

    This file contains DNA oligonucleotides used for the target sequences of tsRNAs and microRNAs in the luciferase vector in Extended Data Fig. 3a.

  8. 8.

    Supplementary Table 6

    This file contains PCR primers for the generation of the Northern probes.

  9. 9.

    Supplementary Table 7

    This file contains Biotin labelled oligonuclotides used for the ChIRP studies in Extended Data Fig. 8a.

  10. 10.

    Supplementary Table 8

    This file contains modified nucleotide sequences of the ribosomal protein mutants. Upper characters are altered sequences and the numbers next to each sequence indicate the sequence position in the each ribosomal protein gene.

  11. 11.

    Supplementary Table 9

    This file contains primers for site-directed mutageneis.

  12. 12.

    Supplementary Table 10

    This file contains icSHAPE scores for the full-length studied mRNAs. Each number represents the scores for each nucleotide. The icSHAPE data are scaled from 0 (no reactivity; double-strandedness) to 1 (maximum reactivity; single-strandedness).

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