Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Exonuclease requirements for mammalian ribosomal RNA biogenesis and surveillance

Nature Structural & Molecular Biology volume 26, pages 490–500 (2019)Cite this article

Subjects

Abstract

Ribosomal RNA (rRNA) biogenesis is a multistep process requiring several nuclear and cytoplasmic exonucleases. The exact processing steps for mammalian 5.8S rRNA remain obscure. Here, using loss-of-function approaches in mouse embryonic stem cells (mESCs) and deep sequencing of rRNA intermediates, we investigate the requirements of exonucleases known to be involved in 5.8S maturation at nucleotide resolution and explore the role of the Perlman syndrome–associated 3′–5′ exonuclease Dis3l2 in rRNA processing. We uncover a novel cytoplasmic intermediate that we name ‘7SB’ rRNA that is generated through sequential processing by distinct exosome complexes. 7SB rRNA can be oligoadenylated by an unknown enzyme and/or oligouridylated by TUT4/7 and subsequently processed by Dis3l2 and Eri1. Moreover, exosome depletion triggers Dis3l2-mediated decay (DMD) as a surveillance pathway for rRNAs. Our data identify previously unknown 5.8S rRNA processing steps and provide nucleotide-level insight into the exonuclease requirements for mammalian rRNA processing.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Analysis of rRNA uridylation in Dis3l2-depleted mESCs.
Fig. 2: 7SB rRNA export and cytoplasmic uridylation.
Fig. 3: Dis3l2-mediated rRNA processing and its relationship with the exosome.
Fig. 4: Deep-sequencing analysis of the 3′ end of 5.8S rRNA species and their precursors after Dis3l2, Exosc3, and Exosc10 perturbation.
Fig. 5: Eri1 exonuclease functions in parallel to Dis3l2 to process uridylated 7SB rRNA.
Fig. 6: Exonuclease requirements for mammalian 5.8S rRNA biogenesis and surveillance.

Similar content being viewed by others

Data availability

Raw sequencing data are deposited with GEO Series accession code GSE129734. Source data for Figs. 1b, 2b, c, e, g, h, 3a, e, f, h, and 5a are available with the paper online. Other data are available upon reasonable request.

References

  1. Ciganda, M. & Williams, N. Eukaryotic 5S rRNA biogenesis. Wiley Inter. Rev. RNA 2, 523–533 (2011).

    Article  CAS  Google Scholar 

  2. Tomecki, R., Sikorski, P. J. & Zakrzewska-Placzek, M. Comparison of preribosomal RNA processing pathways in yeast, plant and human cells - focus on coordinated action of endo- and exoribonucleases. FEBS Lett. 591, 1801–1850 (2017).

    Article  CAS  Google Scholar 

  3. Henras, A. K., Plisson-Chastang, C., O’Donohue, M. F., Chakraborty, A. & Gleizes, P. E. An overview of pre-ribosomal RNA processing in eukaryotes. Wiley Inter. Rev. RNA 6, 225–242 (2015).

    Article  CAS  Google Scholar 

  4. Mullineux, S. T. & Lafontaine, D. L. Mapping the cleavage sites on mammalian pre-rRNAs: where do we stand? Biochimie 94, 1521–1532 (2012).

    Article  CAS  Google Scholar 

  5. Thomson, E. & Tollervey, D. The final step in 5.8S rRNA processing is cytoplasmic in Saccharomyces cerevisiae. Mol. Cell Biol. 30, 976–984 (2010).

    Article  CAS  Google Scholar 

  6. Tafforeau, L. et al. The complexity of human ribosome biogenesis revealed by systematic nucleolar screening of Pre-rRNA processing factors. Mol. Cell 51, 539–551 (2013).

    Article  CAS  Google Scholar 

  7. Chlebowski, A., Lubas, M., Jensen, T. H. & Dziembowski, A. RNA decay machines: the exosome. Biochim Biophys. Acta 1829, 552–560 (2013).

    Article  CAS  Google Scholar 

  8. Liu, Q., Greimann, J. C. & Lima, C. D. Reconstitution, activities, and structure of the eukaryotic RNA exosome. Cell 127, 1223–1237 (2006).

    Article  CAS  Google Scholar 

  9. Makino, D. L., Baumgartner, M. & Conti, E. Crystal structure of an RNA-bound 11-subunit eukaryotic exosome complex. Nature 495, 70–75 (2013).

    Article  CAS  Google Scholar 

  10. Dziembowski, A., Lorentzen, E., Conti, E. & Seraphin, B. A single subunit, Dis3, is essentially responsible for yeast exosome core activity. Nat. Struct. Mol. Biol. 14, 15–22 (2007).

    Article  CAS  Google Scholar 

  11. Allmang, C. et al. The yeast exosome and human PM-Scl are related complexes of 3′ → 5′ exonucleases. Genes Dev. 13, 2148–2158 (1999).

    Article  CAS  Google Scholar 

  12. Mitchell, P., Petfalski, E. & Tollervey, D. The 3′ end of yeast 5.8S rRNA is generated by an exonuclease processing mechanism. Genes Dev. 10, 502–513 (1996).

    Article  CAS  Google Scholar 

  13. Mitchell, P., Petfalski, E., Shevchenko, A., Mann, M. & Tollervey, D. The exosome: a conserved eukaryotic RNA processing complex containing multiple 3′→5′ exoribonucleases. Cell 91, 457–466 (1997).

    Article  CAS  Google Scholar 

  14. Schillewaert, S., Wacheul, L., Lhomme, F. & Lafontaine, D. L. The evolutionarily conserved protein Las1 is required for pre-rRNA processing at both ends of ITS2. Mol. Cell Biol. 32, 430–444 (2012).

    Article  CAS  Google Scholar 

  15. Tomecki, R. et al. The human core exosome interacts with differentially localized processive RNases: hDIS3 and hDIS3L. EMBO J. 29, 2342–2357 (2010).

    Article  CAS  Google Scholar 

  16. Staals, R. H. et al. Dis3-like 1: a novel exoribonuclease associated with the human exosome. EMBO J. 29, 2358–2367 (2010).

    Article  CAS  Google Scholar 

  17. Chang, H. M., Triboulet, R., Thornton, J. E. & Gregory, R. I. A role for the Perlman syndrome exonuclease Dis3l2 in the Lin28-let-7 pathway. Nature 497, 244–248 (2013).

    Article  CAS  Google Scholar 

  18. Lubas, M. et al. Exonuclease hDIS3L2 specifies an exosome-independent 3′-5′ degradation pathway of human cytoplasmic mRNA. EMBO J. 32, 1855–1868 (2013).

    Article  CAS  Google Scholar 

  19. Malecki, M. et al. The exoribonuclease Dis3L2 defines a novel eukaryotic RNA degradation pathway. EMBO J. 32, 1842–1854 (2013).

    Article  CAS  Google Scholar 

  20. Ustianenko, D. et al. Mammalian DIS3L2 exoribonuclease targets the uridylated precursors of let-7 miRNAs. RNA 19, 1632–1638 (2013).

    Article  CAS  Google Scholar 

  21. Triboulet, R., Pirouz, M. & Gregory, R. I. A single let-7 microRNA bypasses LIN28-mediated repression. Cell Rep. 13, 260–266 (2015).

    Article  CAS  Google Scholar 

  22. Pirouz, M. et al. Destabilization of pluripotency in the absence of Mad2l2. Cell Cycle 14, 1596–1610 (2015).

    Article  CAS  Google Scholar 

  23. Astuti, D. et al. Germline mutations in DIS3L2 cause the perlman syndrome of overgrowth and Wilms tumor susceptibility. Nat. Genet 44, 277–284 (2012).

    Article  CAS  Google Scholar 

  24. Pirouz, M., Du, P., Munafo, M. & Gregory, R. I. Dis3l2-mediated decay is a quality control pathway for noncoding RNAs. Cell Rep. 16, 1861–1873 (2016).

    Article  CAS  Google Scholar 

  25. Pirouz, M., Ebrahimi, A. G. & Gregory, R. I. Unraveling 3′-end RNA uridylation at nucleotide resolution. Methods 155, 10–19 (2018).

    Article  Google Scholar 

  26. Ustianenko, D. et al. TUT-DIS3L2 is a mammalian surveillance pathway for aberrant structured non-coding RNAs. EMBO J. 35, 2179–2191 (2016).

    Article  CAS  Google Scholar 

  27. Towler, B. P., Jones, C. I., Harper, K. L., Waldron, J. A. & Newbury, S. F. A novel role for the 3′-5′ exoribonuclease Dis3L2 in controlling cell proliferation and tissue growth. RNA Biol. 13, 1286–1299 (2016).

    Article  Google Scholar 

  28. Reimao-Pinto, M. M. et al. Molecular basis for cytoplasmic RNA surveillance by uridylation-triggered decay in Drosophila. EMBO J. 35, 2417–2434 (2016).

    Article  CAS  Google Scholar 

  29. Labno, A. et al. Perlman syndrome nuclease DIS3L2 controls cytoplasmic non-coding RNAs and provides surveillance pathway for maturing snRNAs. Nucleic Acids Res. 44, 10437–10453 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Eckwahl, M. J., Sim, S., Smith, D., Telesnitsky, A. & Wolin, S. L. A retrovirus packages nascent host noncoding RNAs from a novel surveillance pathway. Genes Dev. 29, 646–657 (2015).

    Article  CAS  Google Scholar 

  31. Preti, M. et al. Gradual processing of the ITS1 from the nucleolus to the cytoplasm during synthesis of the human 18S rRNA. Nucleic Acids Res. 41, 4709–4723 (2013).

    Article  CAS  Google Scholar 

  32. Lim, J. et al. Mixed tailing by TENT4A and TENT4B shields mRNA from rapid deadenylation. Science 361, 701–704 (2018).

    Article  CAS  Google Scholar 

  33. Ansel, K. M. et al. Mouse Eri1 interacts with the ribosome and catalyzes 5.8S rRNA processing. Nat. Struct. Mol. Biol. 15, 523–530 (2008).

    Article  CAS  Google Scholar 

  34. Thornton, J. E., Chang, H. M., Piskounova, E. & Gregory, R. I. Lin28-mediated control of let-7 microRNA expression by alternative TUTases Zcchc11 (TUT4) and Zcchc6 (TUT7). RNA 18, 1875–1885 (2012).

    Article  CAS  Google Scholar 

  35. Thornton, J. E. et al. Selective microRNA uridylation by Zcchc6 (TUT7) and Zcchc11 (TUT4). Nucleic Acids Res. 42, 11777–11791 (2014).

    Article  CAS  Google Scholar 

  36. Hagan, J. P., Piskounova, E. & Gregory, R. I. Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in mouse embryonic stem cells. Nat. Struct. Mol. Biol. 16, 1021–1025 (2009).

    Article  CAS  Google Scholar 

  37. Heo, I. et al. TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation. Cell 138, 696–708 (2009).

    Article  CAS  Google Scholar 

  38. Lin, S., Choe, J., Du, P., Triboulet, R. & Gregory, R. I. The m6A methyltransferase METTL3 promotes translation in human cancer cells. Mol. Cell 62, 335–345 (2016).

    Article  CAS  Google Scholar 

  39. Allmang, C. et al. Functions of the exosome in rRNA, snoRNA and snRNA synthesis. EMBO J. 18, 5399–5410 (1999).

    Article  CAS  Google Scholar 

  40. Hoefig, K. P. et al. Eri1 degrades the stem-loop of oligouridylated histone mRNAs to induce replication-dependent decay. Nat. Struct. Mol. Biol. 20, 73–81 (2013).

    Article  CAS  Google Scholar 

  41. Bowman, L. H., Goldman, W. E., Goldberg, G. I., Hebert, M. B. & Schlessinger, D. Location of the initial cleavage sites in mouse pre-rRNA. Mol. Cell Biol. 3, 1501–1510 (1983).

    Article  CAS  Google Scholar 

  42. Reddy, R. et al. The nucleotide sequence of 8S RNA bound to preribosomal RNA of Novikoff hepatoma. The 5′-end of 8S RNA is 5.8S RNA. J. Biol. Chem. 258, 584–589 (1983).

    CAS  PubMed  Google Scholar 

  43. Michot, B., Joseph, N., Mazan, S. & Bachellerie, J. P. Evolutionarily conserved structural features in the ITS2 of mammalian pre-rRNAs and potential interactions with the snoRNA U8 detected by comparative analysis of new mouse sequences. Nucleic Acids Res. 27, 2271–2282 (1999).

    Article  CAS  Google Scholar 

  44. LaCava, J. et al. RNA degradation by the exosome is promoted by a nuclear polyadenylation complex. Cell 121, 713–724 (2005).

    Article  CAS  Google Scholar 

  45. Chung, C. Z., Jo, D. H. & Heinemann, I. U. Nucleotide specificity of the human terminal nucleotidyltransferase Gld2 (TUT2). RNA 22, 1239–1249 (2016).

    Article  CAS  Google Scholar 

  46. Rammelt, C., Bilen, B., Zavolan, M. & Keller, W. PAPD5, a noncanonical poly(A) polymerase with an unusual RNA-binding motif. RNA 17, 1737–1746 (2011).

    Article  CAS  Google Scholar 

  47. Shcherbik, N., Wang, M., Lapik, Y. R., Srivastava, L. & Pestov, D. G. Polyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells. EMBO Rep. 11, 106–111 (2010).

    Article  CAS  Google Scholar 

  48. Barandun, J., Hunziker, M. & Klinge, S. Assembly and structure of the SSU processome-a nucleolar precursor of the small ribosomal subunit. Curr. Opin. Struct. Biol. 49, 85–93 (2018).

    Article  CAS  Google Scholar 

  49. Cheng, Z. F. & Deutscher, M. P. Quality control of ribosomal RNA mediated by polynucleotide phosphorylase and RNase R. Proc. Natl Acad. Sci. USA 100, 6388–6393 (2003).

    Article  CAS  Google Scholar 

  50. Zhou, X. et al. RdRP-synthesized antisense ribosomal siRNAs silence pre-rRNA via the nuclear RNAi pathway. Nat. Struct. Mol. Biol. 24, 258–269 (2017).

    Article  CAS  Google Scholar 

  51. Dominski, Z., Yang, X. C., Kaygun, H., Dadlez, M. & Marzluff, W. F. A 3′ exonuclease that specifically interacts with the 3′ end of histone mRNA. Mol. Cell 12, 295–305 (2003).

    Article  CAS  Google Scholar 

  52. Yang, X. C., Purdy, M., Marzluff, W. F. & Dominski, Z. Characterization of 3′hExo, a 3′ exonuclease specifically interacting with the 3′ end of histone mRNA. J. Biol. Chem. 281, 30447–30454 (2006).

    Article  CAS  Google Scholar 

  53. Gabel, H. W. & Ruvkun, G. The exonuclease ERI-1 has a conserved dual role in 5.8S rRNA processing and RNAi. Nat. Struct. Mol. Biol. 15, 531–533 (2008).

    Article  CAS  Google Scholar 

  54. Kennedy, S., Wang, D. & Ruvkun, G. A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans. Nature 427, 645–649 (2004).

    Article  CAS  Google Scholar 

  55. Tan, D., Marzluff, W. F., Dominski, Z. & Tong, L. Structure of histone mRNA stem-loop, human stem-loop binding protein, and 3′hExo ternary complex. Science 339, 318–321 (2013).

    Article  CAS  Google Scholar 

  56. Cote, C. A., Greer, C. L. & Peculis, B. A. Dynamic conformational model for the role of ITS2 in pre-rRNA processing in yeast. RNA 8, 786–797 (2002).

    Article  CAS  Google Scholar 

  57. Joseph, N., Krauskopf, E., Vera, M. I. & Michot, B. Ribosomal internal transcribed spacer 2 (ITS2) exhibits a common core of secondary structure in vertebrates and yeast. Nucleic Acids Res. 27, 4533–4540 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants to R.I.G. from the US National Institutes of Health (R01GM086386; R01CA211328) and the March of Dimes Foundation (FY15-3339). M.P. was supported by a research fellowship from the Manton Center for Orphan Disease Research.

Author information

Authors and Affiliations

  1. Stem Cell Program, Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA, USA

    Mehdi Pirouz, Marzia Munafò, Junho Choe & Richard I. Gregory

  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Mehdi Pirouz, Junho Choe & Richard I. Gregory

  3. The Manton Center for Orphan Disease Research, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

    Mehdi Pirouz

  4. Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Harvard Medical School, Boston, MA, USA

    Aref G. Ebrahimi

  5. Department of Life Science, College of Natural Sciences, Research Institute for Natural Sciences, Hanyang University, Seoul, Republic of Korea

    Junho Choe

  6. Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    Richard I. Gregory

  7. Harvard Initiative for RNA Medicine, Boston, MA, USA

    Richard I. Gregory

  8. Harvard Stem Cell Institute, Cambridge, MA, USA

    Richard I. Gregory

Authors
  1. Mehdi Pirouz
    View author publications

    Search author on:PubMed Google Scholar

  2. Marzia Munafò
    View author publications

    Search author on:PubMed Google Scholar

  3. Aref G. Ebrahimi
    View author publications

    Search author on:PubMed Google Scholar

  4. Junho Choe
    View author publications

    Search author on:PubMed Google Scholar

  5. Richard I. Gregory
    View author publications

    Search author on:PubMed Google Scholar

Contributions

M.P. performed all experiments with help from M.M. and J.C. A.G.E. and M.P. performed the bioinformatics analysis. M.P., M.M., and R.I.G. designed all experiments, analyzed data, and wrote the manuscript.

Corresponding author

Correspondence to Richard I. Gregory.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Integrated supplementary information

Supplementary Figure 1 Role of Dis3l2 in 7SB rRNA processing.

(a) Western blot analysis of Dis3l2 expression in wild type (WT), heterozygous (Het), and knockout (KO) mESC lines used in this study. (b) Probing 7SB rRNA in Dis3l2 stable knockdown V6.5 mESC line. (c) qRT-PCR analysis of relative uridylation for indicated mature or intermediate rRNA species in mESCs stably transduced with shLacZ (as control) and shDis3l2 shRNAs. qRT-PCR (d) and northern blot (e) analyses of FLAG-mutant Dis3l2-bound 5.8S rRNA with indicated primer sets or probe, respectively. Western blot (f) and qRT-PCR (g) indicate that 7SB uridylation can be rescued by FLAG-wild type Dis3l2 re-expression. Bars represent mean ± SEM; *p ≤ 0.05,**p ≤ 0.01, ***p ≤ 0.001, Student’s t-test (n ≥ 3); ns, not significant. Uncropped blot/gel images are shown in Supplementary Data Set 1.

Supplementary Figure 2 Sequence analysis of 3′-ends of Dis3l2-targetted 5.8S rRNA.

(a) Schematic representation of circular RACE (cRACE) protocol to study 7SB rRNA tails. Total RNAs from mutant FLAG-Dis3l2 RIP and input samples were isolated, and treated with T4 RNA ligase. After DNase I treatment, reverse transcription was performed using reverse primer (overlapping 5.8S rRNA and ITS2) to enrich for 7SB species. PCR with divergent forward and reverse primers generated products that were purified and sequenced by MiSeq (see methods section for details). (b) Pie chart representation of various tails in extended 5.8S in RIP samples showed that tails contain mostly U or both A and then U (purple). Only a small set of the extended reads have A-tail but no U-tail (blue). (c) Size distribution of extensions (Ext.), A- and U-tails in RIP samples. (d) qRT-PCR analysis of 5.8S rRNA levels in Dis3l2 depleted mESCs. (e) Left panel: human DIS3L2 mRNA expression in stable knockdown human cell lines. Relative uridylation levels of human 7SB rRNA (middle panel) and pre-let-7a1 miRNA (right panel as a positive control) are measured by qRT-PCR. Bars represent mean ± SEM; **p ≤ 0.01, ***p ≤ 0.001, Student’s t-test (n ≥ 3); ns, not significant.

Supplementary Figure 3 Dis3l2 re-expression attenuates uridylation of pre-rRNAs stabilized by Exosc3- and Exosc10-depletion.

(a) Left panel: qRT-PCR analysis shows specific knockdown of Exosc3 and Exosc10 using specific siRNAs in Dis3l2 knockout ESCs. Right panel: qRT-PCR analysis revealing the rescue effect of Dis3l2 on uridylation of rRNAs. 7SK uridylation was unchanged, whereas Rmrp uridylation was only rescued by Dis3l2 re-expression but not induced upon Exosc3- or Exosc10-depletion.

Supplementary information

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pirouz, M., Munafò, M., Ebrahimi, A.G. et al. Exonuclease requirements for mammalian ribosomal RNA biogenesis and surveillance. Nat Struct Mol Biol 26, 490–500 (2019). https://doi.org/10.1038/s41594-019-0234-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41594-019-0234-x

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing