Article | Published:

Exonuclease requirements for mammalian ribosomal RNA biogenesis and surveillance


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.

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.


  1. 1.

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

  2. 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).

  3. 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).

  4. 4.

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

  5. 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).

  6. 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).

  7. 7.

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

  8. 8.

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

  9. 9.

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

  10. 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).

  11. 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).

  12. 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).

  13. 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).

  14. 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).

  15. 15.

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

  16. 16.

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

  17. 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).

  18. 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).

  19. 19.

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

  20. 20.

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

  21. 21.

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

  22. 22.

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

  23. 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).

  24. 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).

  25. 25.

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

  26. 26.

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

  27. 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).

  28. 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).

  29. 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).

  30. 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).

  31. 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).

  32. 32.

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

  33. 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).

  34. 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).

  35. 35.

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

  36. 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).

  37. 37.

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

  38. 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).

  39. 39.

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

  40. 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).

  41. 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).

  42. 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).

  43. 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).

  44. 44.

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

  45. 45.

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

  46. 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).

  47. 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).

  48. 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).

  49. 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).

  50. 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).

  51. 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).

  52. 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).

  53. 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).

  54. 54.

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

  55. 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).

  56. 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).

  57. 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).

Download references


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

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.

Competing interests

The authors declare no competing interests.

Correspondence to Richard I. Gregory.

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

Supplementary Information

Supplementary Figures 1–3, Supplementary Dataset 1

Reporting Summary

Supplementary Table 1

Source data

Source Data Fig. 1

Source Data Fig. 2

Source Data Fig. 3

Source Data Fig. 5

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark
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.
Supplementary Figure 1: Role of Dis3l2 in 7SB rRNA processing.
Supplementary Figure 2: Sequence analysis of 3′-ends of Dis3l2-targetted 5.8S rRNA.
Supplementary Figure 3: Dis3l2 re-expression attenuates uridylation of pre-rRNAs stabilized by Exosc3- and Exosc10-depletion.