Skip to main content

Thank you for visiting 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.

Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits


In the final steps of yeast ribosome synthesis, immature translation-incompetent pre-40S particles that contain 20S pre-rRNA are converted to the mature translation-competent subunits containing the 18S rRNA. An assay for 20S pre-rRNA cleavage in purified pre-40S particles showed that cleavage by the PIN domain endonuclease Nob1 was strongly stimulated by the GTPase activity of Fun12, the yeast homolog of cytoplasmic translation initiation factor eIF5b. Cleavage of the 20S pre-rRNA was also inhibited in vivo and in vitro by blocking binding of Fun12 to the 25S rRNA through specific methylation of its binding site. Cleavage competent pre-40S particles stably associated with Fun12 and formed 80S complexes with 60S ribosomal subunits. We propose that recruitment of 60S subunits promotes GTP hydrolysis by Fun12, leading to structural rearrangements within the pre-40S particle that bring Nob1 and the pre-rRNA cleavage site together.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: In vitro RNA cleavage by Nob1 is not affected by sequences 3′ to site A2.
Figure 2: In vitro cleavage in pre-40S particles is stimulated by ATP or GTP addition.
Figure 3: Fun12 is associated with pre-rRNA.
Figure 4: Fun12 is required for efficient pre-20S pre-rRNA processing.
Figure 5: Fun12 is responsible for GTP-mediated stimulation of in vitro cleavage.
Figure 6: Fun12 binding to 25S rRNA is required for efficient 20S processing.
Figure 7: Pre-40S particles stably associate with Fun12 and mature 60S particles before final 40S particle maturation.
Figure 8: Model for the role of Fun12 in pre-40S processing.

Accession codes


NCBI Reference Sequence


  1. 1

    Inoue, K., Alsina, J., Chen, J. & Inouye, M. Suppression of defective ribosome assembly in a rbfA deletion mutant by overexpression of Era, an essential GTPase in Escherichia coli. Mol. Microbiol. 48, 1005–1016 (2003).

    CAS  Article  Google Scholar 

  2. 2

    Granneman, S., Petfalski, E., Swiatkowska, A. & Tollervey, D. Cracking pre-40S ribosomal subunit structure by systematic analyses of RNA-protein cross-linking. EMBO J. 29, 2026–2036 (2010).

    CAS  Article  Google Scholar 

  3. 3

    Li, Z. et al. Rational extension of the ribosome biogenesis pathway using network-guided genetics. PLoS Biol. 7, e1000213 (2009).

    Article  Google Scholar 

  4. 4

    Campbell, T.L. & Brown, E.D. Genetic interaction screens with ordered overexpression and deletion clone sets implicate the Escherichia coli GTPase YjeQ in late ribosome biogenesis. J. Bacteriol. 190, 2537–2545 (2008).

    CAS  Article  Google Scholar 

  5. 5

    Lo, K.Y. et al. Defining the pathway of cytoplasmic maturation of the 60S ribosomal subunit. Mol. Cell 39, 196–208 (2010).

    CAS  Article  Google Scholar 

  6. 6

    Sengupta, J. et al. Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit. J. Cell Biol. 189, 1079–1086 (2010).

    CAS  Article  Google Scholar 

  7. 7

    Kemmler, S., Occhipinti, L., Veisu, M. & Panse, V.G. Yvh1 is required for a late maturation step in the 60S biogenesis pathway. J. Cell Biol. 186, 863–880 (2009).

    CAS  Article  Google Scholar 

  8. 8

    Strunk, B.S. et al. Ribosome assembly factors prevent premature translation initiation by 40S assembly intermediates. Science 333, 1449–1453 (2011).

    CAS  Article  Google Scholar 

  9. 9

    Panse, V.G. & Johnson, A.W. Maturation of eukaryotic ribosomes: acquisition of functionality. Trends Biochem. Sci. 35, 260–266 (2010).

    CAS  Article  Google Scholar 

  10. 10

    Soudet, J., Gelugne, J.P., Belhabich-Baumas, K., Caizergues-Ferrer, M. & Mougin, A. Immature small ribosomal subunits can engage in translation initiation in Saccharomyces cerevisiae. EMBO J. 29, 80–92 (2010).

    CAS  Article  Google Scholar 

  11. 11

    Pertschy, B. et al. RNA helicase Prp43 and its co-factor Pfa1 promote 20 to 18 S rRNA processing catalyzed by the endonuclease Nob1. J. Biol. Chem. 284, 35079–35091 (2009).

    CAS  Article  Google Scholar 

  12. 12

    Fatica, A., Oeffinger, M., Dlakic, M. & Tollervey, D. Nob1p is required for cleavage of the 3′ end of 18S rRNA. Mol. Cell. Biol. 23, 1798–1807 (2003).

    CAS  Article  Google Scholar 

  13. 13

    Fatica, A., Tollervey, D. & Dlakic, M. The PIN domain of Nob1p is required for 20S pre-rRNA cleavage at site D. RNA 10, 1698–1701 (2004).

    CAS  Article  Google Scholar 

  14. 14

    Lamanna, A.C. & Karbstein, K. Nob1 binds the single-stranded cleavage site D at the 3′-end of 18S rRNA with its PIN domain. Proc. Natl. Acad. Sci. USA 106, 14259–14264 (2009).

    CAS  Article  Google Scholar 

  15. 15

    Woolls, H.A., Lamanna, A.C. & Karbstein, K. Roles of Dim2 in ribosome assembly. J. Biol. Chem. 286, 2578–2586 (2011).

    CAS  Article  Google Scholar 

  16. 16

    Lebaron, S. et al. The ATPase and helicase activities of Prp43p are stimulated by the G-patch protein Pfa1p during yeast ribosome biogenesis. EMBO J. 28, 3808–3819 (2009).

    CAS  Article  Google Scholar 

  17. 17

    Lamanna, A.C. & Karbstein, K. An RNA conformational switch regulates pre-18S rRNA cleavage. J. Mol. Biol. 405, 3–17 (2011).

    CAS  Article  Google Scholar 

  18. 18

    Allmang, C. et al. Processing of the yeast pre-rRNA at sites A2 and A3 is linked. RNA 2, 63–73 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Yeh, L.-C.C., Thweatt, R. & Lee, J.C. Internal transcribed spacer 1 of the yeast precursor ribosomal RNA. Higher order structure and common structural motifs. Biochemistry 29, 5911–5918 (1990).

    CAS  Article  Google Scholar 

  20. 20

    Van Nues, R.W. et al. Separate structural elements within internal transcribed spacer 1 of Saccharomyces cerevisiae precursor ribosomal RNA direct the formation of 17S and 26S rRNA. Nucleic Acids Res. 22, 912–919 (1994).

    CAS  Article  Google Scholar 

  21. 21

    Gelperin, D., Horton, L., Beckman, J., Hensold, J. & Lemmon, S.K. Bms1p, a novel GTP-binding protein, and the related Tsr1p are required for distinct steps of 40S ribosome biogenesis in yeast. RNA 7, 1268–1283 (2001).

    CAS  Article  Google Scholar 

  22. 22

    Lee, J.H. et al. Initiation factor eIF5B catalyzes second GTP-dependent step in eukaryotic translation initiation. Proc. Natl. Acad. Sci. USA 99, 16689–16694 (2002).

    CAS  Article  Google Scholar 

  23. 23

    Pestova, T.V. et al. The joining of ribosomal subunits in eukaryotes requires eIF5B. Nature 403, 332–335 (2000).

    CAS  Article  Google Scholar 

  24. 24

    Ferreira-Cerca, S. et al. Analysis of the in vivo assembly pathway of eukaryotic 40S ribosomal proteins. Mol. Cell 28, 446–457 (2007).

    CAS  Article  Google Scholar 

  25. 25

    Udem, S.A. & Warner, J.R. The cytoplasmic maturation of a ribosomal precursor ribonucleic acid in yeast. J. Biol. Chem. 248, 1412–1416 (1973).

    CAS  PubMed  Google Scholar 

  26. 26

    Brand, R.C., Klootwijk, J., van Steenbergen, T.J.M., de Kok, A.J. & Planta, R.J. Secondary methylation of yeast ribosomal precursor RNA. Eur. J. Biochem. 75, 311–318 (1977).

    CAS  Article  Google Scholar 

  27. 27

    Lafontaine, D., Delcour, J., Glasser, A.-L., Desgrès, J. & Vandenhaute, J. The DIM1 gene responsible for the conserved m26Am26A dimethylation in the 3′-terminal loop of 18S rRNA is essential in yeast. J. Mol. Biol. 241, 492–497 (1994).

    CAS  Article  Google Scholar 

  28. 28

    van Nues, R.W. et al. Box C/D snoRNP catalysed methylation is aided by additional pre-rRNA base-pairing. EMBO J. 30, 2420–2430 (2011).

    CAS  Article  Google Scholar 

  29. 29

    Unbehaun, A. et al. Position of eukaryotic initiation factor eIF5B on the 80S ribosome mapped by directed hydroxyl radical probing. EMBO J. 26, 3109–3123 (2007).

    CAS  Article  Google Scholar 

  30. 30

    Shin, B.-S. et al. rRNA suppressor of a eukaryotic translation initiation factor 5B/Initiation factor 2 mutant reveals a binding site for translational GTPases on the small ribosomal subunit. Mol. Cell. Biol. 29, 808–821 (2009).

    CAS  Article  Google Scholar 

  31. 31

    Decatur, W.A. & Fournier, M.J. rRNA modifications and ribosome function. Trends Biochem. Sci. 27, 344–351 (2002).

    CAS  Article  Google Scholar 

  32. 32

    Lygerou, Z., Allmang, C., Tollervey, D. & Séraphin, B. Accurate processing of a eukaryotic pre-rRNA by RNase MRP in vitro. Science 272, 268–270 (1996).

    CAS  Article  Google Scholar 

  33. 33

    Abou Elela, S., Igel, H. & Ares, M. Jr. RNase III cleaves eukaryotic preribosomal RNA at a U3 snoRNP-dependent site. Cell 85, 115–124 (1996).

    Article  Google Scholar 

  34. 34

    Horn, D.M., Mason, S.L. & Karbstein, K. Rcl1 protein, a novel nuclease for 18 S ribosomal RNA production. J. Biol. Chem. 286, 34082–34087 (2011).

    CAS  Article  Google Scholar 

  35. 35

    Allmang, C. & Tollervey, D. The role of the 3′ external transcribed spacer in yeast pre-rRNA processing. J. Mol. Biol. 278, 67–78 (1998).

    CAS  Article  Google Scholar 

  36. 36

    Valasek, L., Hasek, J., Nielsen, K.H. & Hinnebusch, A.G. Dual function of eIF3j/Hcr1p in processing 20 S pre-rRNA and translation initiation. J. Biol. Chem. 276, 43351–43360 (2001).

    CAS  Article  Google Scholar 

  37. 37

    Collins, S.R. et al. Toward a comprehensive atlas of the physical interactome of Saccharomyces cerevisiae. Mol. Cell. Proteomics 6, 439–450 (2007).

    CAS  Article  Google Scholar 

  38. 38

    Senger, B. et al. The nucle(ol)ar Tif6p and Efl1p are required for a late cytoplasmic step of ribosome synthesis. Mol. Cell 8, 1363–1373 (2001).

    CAS  Article  Google Scholar 

  39. 39

    Yarunin, A. et al. Functional link between ribosome formation and biogenesis of iron-sulfur proteins. EMBO J. 24, 580–588 (2005).

    CAS  Article  Google Scholar 

  40. 40

    Neueder, A. et al. A local role for the small ribosomal subunit primary binder rpS5 in final 18S rRNA processing in yeast. PLoS ONE 5, e10194 (2010).

    Article  Google Scholar 

  41. 41

    Veith, T. et al. Structural and functional analysis of the archaeal endonuclease Nob1. Nucleic Acids Res. 40, 3259–3274 (2012).

    CAS  Article  Google Scholar 

  42. 42

    Marshall, R.A., Aitken, C.E. & Puglisi, J.D. GTP hydrolysis by IF2 guides progression of the ribosome into elongation. Mol. Cell 35, 37–47 (2009).

    CAS  Article  Google Scholar 

  43. 43

    Julian, P. et al. The cryo-EM structure of a complete 30S translation initiation complex from Escherichia coli. PLoS Biol. 9, e1001095 (2011).

    CAS  Article  Google Scholar 

  44. 44

    La Teana, A., Gualerzi, C.O. & Brimacombe, R. From stand-by to decoding site. Adjustment of the mRNA on the 30S ribosomal subunit under the influence of the initiation factors. RNA 1, 772–782 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Valle, M. et al. Locking and unlocking of ribosomal motions. Cell 114, 123–134 (2003).

    CAS  Article  Google Scholar 

  46. 46

    Mangiarotti, G., Chiaberge, S. & Bulfone, S. rRNA maturation as a “quality” control step in ribosomal subunit assembly in Dictyostelium discoideum. J. Biol. Chem. 272, 27818–27822 (1997).

    CAS  Article  Google Scholar 

  47. 47

    Kaczanowska, M. & Ryden-Aulin, M. Ribosome biogenesis and the translation process in Escherichia coli. Microbiol. Mol. Biol. Rev. 71, 477–494 (2007).

    CAS  Article  Google Scholar 

  48. 48

    Belotserkovsky, J.M., Dabbs, E.R. & Isaksson, L.A. Mutations in 16S rRNA that suppress cold-sensitive initiation factor 1 affect ribosomal subunit association. FEBS J. 278, 3508–3517 (2011).

    CAS  Article  Google Scholar 

  49. 49

    Ben-Shem, A. et al. The structure of the eukaryotic ribosome at 3.0 A resolution. Science 334, 1524–1529 (2011).

    CAS  Article  Google Scholar 

  50. 50

    Brachmann, C.B. et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132 (1998).

    CAS  Article  Google Scholar 

  51. 51

    Petracek, M.E. & Longtine, M.S. PCR-based engineering of yeast genome. Methods Enzymol. 350, 445–469 (2002).

    CAS  Article  Google Scholar 

  52. 52

    Bohnsack, M.T. et al. Prp43 bound at different sites on the pre-rRNA performs distinct functions in ribosome synthesis. Mol. Cell 36, 583–592 (2009).

    CAS  Article  Google Scholar 

  53. 53

    Karbstein, K., Jonas, S. & Doudna, J.A. An essential GTPase promotes assembly of preribosomal RNA processing complexes. Mol. Cell 20, 633–643 (2005).

    CAS  Article  Google Scholar 

  54. 54

    Granneman, S., Kudla, G., Petfalski, E. & Tollervey, D. Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high throughput analysis of cDNAs. Proc. Natl. Acad. Sci. USA 106, 9613–9618 (2009).

    CAS  Article  Google Scholar 

  55. 55

    Lebaron, S. et al. The splicing ATPase prp43p is a component of multiple preribosomal particles. Mol. Cell. Biol. 25, 9269–9282 (2005).

    CAS  Article  Google Scholar 

  56. 56

    Tollervey, D. High level of complexity of small nuclear RNAs from fungi and plants. J. Mol. Biol. 196, 355–361 (1987).

    CAS  Article  Google Scholar 

  57. 57

    Leshin, J.A., Rakauskaite, R., Dinman, J.D. & Meskauskas, A. Enhanced purity, activity and structural integrity of yeast ribosomes purified using a general chromatographic method. RNA Biol. 7, 354–360 (2010).

    CAS  Article  Google Scholar 

  58. 58

    Roll-Mecak, A., Cao, C., Dever, T.E. & Burley, S.K. X-ray structures of the universal translation initiation factor IF2/eIF5B: conformational changes on GDP and GTP binding. Cell 103, 781–792 (2000).

    CAS  Article  Google Scholar 

Download references


This work was supported by the Wellcome Trust (S.L., C.S., S.G. and D.T., 077248; R.W.v.N. and N.J.W., WT089378MA), the Royal Society (C.S.), The Darwin Trust of Edinburgh (B.B.) and an European Molecular Biology Organisation Long-Term Fellowship (S.L.). The electron microscopy facility was supported by the Wellcome Trust and the Scottish Universities Life Sciences Alliance. Work in the Wellcome Trust Centre for Cell Biology is supported by Wellcome Trust core funding (092076). We thank E. Fayet-Lebaron for her technical support and critical reading of the manuscript. We thank members of the Edinburgh Protein Production Facility for protein purification. Use of the Edinburgh Protein Production Facility was supported by The Wellcome Trust, the Scottish Universities Life Sciences Alliance and the Biotechnology and Biological Sciences Research Council.

Author information




S.L., C.S., R.W.v.N., A.S., S.G., N.J.W. and D.T. designed experiments; S.L., C.S., R.W.v.N., A.S., D.W., B.B. and S.G. performed experiments; S.L., C.S., R.W.v.N., A.S., B.B., S.G., N.J.W. and D.T. analyzed data; and S.L., C.S., R.W.v.N., A.S., S.G., N.J.W. and D.T. wrote the paper.

Corresponding author

Correspondence to David Tollervey.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1–2 and Supplementary Note (PDF 9758 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lebaron, S., Schneider, C., van Nues, R. et al. Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits. Nat Struct Mol Biol 19, 744–753 (2012).

Download citation

Further reading


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