Location of Repository

The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site

By Alexander B. Taylor, Britta Meyer, Belinda Z. Leal, Peter Kötter, Virgil Schirf, Borries Demeler, P. John Hart, Karl-Dieter Entian and Jens Wöhnert

Abstract

Ribosome biogenesis in eukaryotes requires the participation of a large number of ribosome assembly factors. The highly conserved eukaryotic nucleolar protein Nep1 has an essential but unknown function in 18S rRNA processing and ribosome biogenesis. In Saccharomyces cerevisiae the malfunction of a temperature-sensitive Nep1 protein (nep1-1ts) was suppressed by the addition of S-adenosylmethionine (SAM). This suggests the participation of Nep1 in a methyltransferase reaction during ribosome biogenesis. In addition, yeast Nep1 binds to a 6-nt RNA-binding motif also found in 18S rRNA and facilitates the incorporation of ribosomal protein Rps19 during the formation of pre-ribosomes. Here, we present the X-ray structure of the Nep1 homolog from the archaebacterium Methanocaldococcus jannaschii in its free form (2.2 Å resolution) and bound to the S-adenosylmethionine analog S-adenosylhomocysteine (SAH, 2.15 Å resolution) and the antibiotic and general methyltransferase inhibitor sinefungin (2.25 Å resolution). The structure reveals a fold which is very similar to the conserved core fold of the SPOUT-class methyltransferases but contains a novel extension of this common core fold. SAH and sinefungin bind to Nep1 at a preformed binding site that is topologically equivalent to the cofactor-binding site in other SPOUT-class methyltransferases. Therefore, our structures together with previous genetic data suggest that Nep1 is a genuine rRNA methyltransferase

Topics: Structural Biology
Publisher: Oxford University Press
OAI identifier: oai:pubmedcentral.nih.gov:2275143
Provided by: PubMed Central
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://www.pubmedcentral.nih.g... (external link)
  • Suggested articles

    Preview

    Citations

    1. (1999). A computational screen for methylation guide snoRNAs in yeast.
    2. (2004). A surfeit of factors: why is ribosome assembly so much more complicated in eukaryotes than bacteria?
    3. (1994). An automated procedure for phase improvement by density modification.
    4. (2002). An enzyme with a deep trefoil knot for the active-site architecture.
    5. (1993). Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin.
    6. (2006). Automated structure solution with autoSHARP.
    7. (2004). Coot: model-building tools for molecular graphics.
    8. (2006). Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements.
    9. (2000). Crystal structure of a fibrillarin homologue from Methanococcus jannaschii, a hyperthermophile, at 1.6A ˚ resolution.
    10. (2003). Crystal structure of tRNA(m1G37)methyltransferase: insights into tRNA recognition.
    11. (2004). Deep knot structure for construction of active site and cofactor binding site of tRNA modification enzyme.
    12. (2003). Diversity of proteinprotein interactions.
    13. (2003). Functional assignment based on structural analysis: crystal structure of the yggJ protein (HI0303) of Haemophilus influenzae reveals an RNA methyltransferase with a deep trefoil knot.
    14. (2006). Genetic evidence for 18S rRNA binding and an Rps19p assembly function of yeast nucleolar protein Nep1p.
    15. (2006). Identification and characterization of RsmE, the founding member of a new RNA base methyltransferase family.
    16. (1996). In vitro complementation analysis localizes 23S rRNA posttranscriptional modifications that are required for Escherichia coli 50S ribosomal subunit assembly and function.
    17. (2003). Insights into catalysis by a knotted TrmD tRNA methyltransferase.
    18. (1997). Maximum-likelihood heavy-atom parameter refinement for the multiple isomorphous replacement and multiwavelength anomalous diffraction methods.
    19. (2004). Mechanism of RNA 20-O-methylation: evidence that the catalytic lysine acts to steer rather than deprotonate the target nucleophile.
    20. (2007). Monte Carlo analysis of sedimentation experiments.
    21. (2002). Nep1p (Emg1p), a novel protein conserved in eukaryotes and archaea, is involved in ribosome biogenesis.
    22. (2001). Novel stress-responsive genes EMG1 and NOP14 encode conserved, interacting proteins required for 40S ribosome biogenesis.
    23. (2002). PHENIX: building new software for automated crystallographic structure determination.
    24. (1995). Posttranscriptional modification of the central loop of domain V in Escherichia coli 23S ribosomal RNA.
    25. (1998). Posttranscriptional modifications in 16S and 23S rRNAs of the archaeal hyperthermophile Sulfolobus solfataricus.
    26. (1997). Processing of X-ray diffraction data collected in oscillation mode.
    27. (2003). Ribosome assembly in eukaryotes.
    28. (2002). Role of entropy in protein thermostability: folding kinetics of a hyperthermophilic cold shock protein at high temperatures using 19F NMR.
    29. (2005). Roles of conserved amino acid sequence motifs in the SpoU (TrmH) RNA methyltransferase family.
    30. (2002). rRNA modifications and ribosome function.
    31. (2002). SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold.
    32. (1996). Site-specific ribose methylation of preribosomal RNA: a novel function for small nucleolar RNAs.
    33. (2004). Spb1p-directed formation of Gm2922 in the ribosome catalytic center occurs at a late processing stage.
    34. (2002). SPOUT: a class of methyltransferases that includes spoU and trmD RNA methylase superfamilies, and novel superfamilies of predicted prokaryotic RNA methylases.
    35. (2007). Structural and evolutionary bioinformatics of the SPOUT superfamily of methyltransferases.
    36. (2005). Structure and function of the antibiotic resistance mediating methyltransferase AviRb from Streptomyces viridochromogenes.
    37. (1999). Structure and function of the nucleolus.
    38. (2004). Structure and mechanism of mRNA cap (guanine-N7) methyltransferase.
    39. (2005). Structure of a class II TrmH tRNA-modifying enzyme from Aquifex aeolicus.
    40. (2000). Structure of functionally activated small ribosomal subunit at 3.3 angstroms resolution.
    41. (2000). Structure of the 30S ribosomal subunit.
    42. (2006). Structure of the 70S ribosome complexed with mRNA and tRNA.
    43. (2001). Structure of the 80S ribosome from Saccharomyces cerevisiae -tRNA-ribosome and subunit-subunit interactions.
    44. (2005). Structures of the bacterial ribosome at 3.5A ˚ resolution.
    45. (1993). Temperature-sensitive mutations demonstrate roles for yeast fibrillarin in pre-rRNA processing, pre-rRNA methylation, and ribosome assembly.
    46. (2001). The ARP/wARP suite for automated construction and refinement of protein models.
    47. (1998). The box H + ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase.
    48. (2000). The complete atomic structure of the large ribosomal subunit at
    49. (1994). The DIM1 gene responsible for the conserved m6(2)Am6(2)A dimethylation in the 30-terminal loop of 18S rRNA is essential in yeast.
    50. (1999). The economics of ribosome biosynthesis in yeast.
    51. (1999). The finer things in X-ray diffraction data collection.
    52. (1995). The nucleolus: an organelle formed by the act of building a ribosome.
    53. (2002). The PyMOL Molecular Graphics System. DeLano Scientific,
    54. (1996). The RNA world of the nucleolus: two major families of small RNAs defined by different box elements with related functions.
    55. (2002). The structure of the RlmB 23S rRNA methyltransferase reveals a new methyltransferase fold with a unique knot.
    56. (2005). UltraScan: a comprehensive data analysis software package for analytical ultracentrifugation experiments.

    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.