Purified Box C/D snoRNPs Are Able To Reproduce Site-Specific 2′-O-Methylation of Target RNA In Vitro

ABSTRACT Small nucleolar RNAs (snoRNAs) are associated in ribonucleoprotein particles localized to the nucleolus (snoRNPs). Most of the members of the box C/D family function in directing site-specific 2′-O-methylation of substrate RNAs. Although the selection of the target nucleotide requires the antisense element and the conserved box D or D′ of the snoRNA, the methyltransferase activity is supposed to reside in one of the protein components. Through protein tagging of a snoRNP-specific factor, we purified to homogeneity box C/D snoRNPs from the yeast Saccharomyces cerevisiae. Mass spectrometric analysis demonstrated the presence of Nop1p, Nop58p, Nop56p, and Snu13p as integral components of the particle. We show that purified snoRNPs are able to reproduce the site-specific methylation pattern on target RNA and that the predicted S-adenosyl-l-methionine-binding region of Nop1p is responsible for the catalytic activity.

[1]  Enzymatic 2′‐O‐methylation of the wobble nucleoside of eukaryotic tRNAPhe: specificity depends on structural elements outside the anticodon loop. , 1986, The EMBO journal.

[2]  D. Tollervey,et al.  A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability. , 1989, The EMBO journal.

[3]  K. Watanabe,et al.  Recognition sites of tRNA by a thermostable tRNA(guanosine-2'-)-methyltransferase from Thermus thermophilus HB27. , 1990, Journal of biochemistry.

[4]  D. Tollervey,et al.  Temperature-sensitive mutations demonstrate roles for yeast fibrillarin in pre-rRNA processing, pre-rRNA methylation, and ribosome assembly , 1993, Cell.

[5]  M. Fournier,et al.  The small nucleolar RNAs. , 1995, Annual review of biochemistry.

[6]  E V Koonin,et al.  Sequence similarity analysis of Escherichia coli proteins: functional and evolutionary implications. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[7]  J. Bachellerie,et al.  Novel intron-encoded small nucleolar RNAs with long sequence complementarities to mature rRNAs involved in ribosome biogenesis. , 1995, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[8]  Tamás Kiss,et al.  Site-Specific Ribose Methylation of Preribosomal RNA: A Novel Function for Small Nucleolar RNAs , 1996, Cell.

[9]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

[10]  D. Tollervey,et al.  Function and synthesis of small nucleolar RNAs. , 1997, Current opinion in cell biology.

[11]  J. Steitz,et al.  Sno Storm in the Nucleolus: New Roles for Myriad Small RNPs , 1997, Cell.

[12]  J. Bachellerie,et al.  Guiding ribose methylation of rRNA. , 1997, Trends in biochemical sciences.

[13]  D. Tollervey,et al.  Nucleolar KKE/D repeat proteins Nop56p and Nop58p interact with Nop1p and are required for ribosome biogenesis , 1997, Molecular and cellular biology.

[14]  J. Brockenbrough,et al.  Nop5p Is a Small Nucleolar Ribonucleoprotein Component Required for Pre-18 S rRNA Processing in Yeast* , 1998, The Journal of Biological Chemistry.

[15]  J. Steitz,et al.  Modification of U6 spliceosomal RNA is guided by other small RNAs. , 1998, Molecular cell.

[16]  D. Tollervey,et al.  Nop58p is a common component of the box C+D snoRNPs that is required for snoRNA stability. , 1999, RNA.

[17]  W. Filipowicz,et al.  Structure and biogenesis of small nucleolar RNAs acting as guides for ribosomal RNA modification. , 1999, Acta biochimica Polonica.

[18]  S. Clarke,et al.  S-Adenosylmethionine-dependent Methylation in Saccharomyces cerevisiae , 1999, The Journal of Biological Chemistry.

[19]  B. Séraphin,et al.  A generic protein purification method for protein complex characterization and proteome exploration , 1999, Nature Biotechnology.

[20]  J. Steitz,et al.  Guided tours: from precursor snoRNA to functional snoRNP. , 1999, Current opinion in cell biology.

[21]  E. Maxwell,et al.  Box C/D snoRNA-associated proteins: two pairs of evolutionarily ancient proteins and possible links to replication and transcription. , 2000, RNA.

[22]  S. Eddy,et al.  Homologs of small nucleolar RNAs in Archaea. , 2000, Science.

[23]  A. Fatica,et al.  Yeast snoRNA accumulation relies on a cleavage‐dependent/polyadenylation‐independent 3′‐processing apparatus , 2000, The EMBO journal.

[24]  U. Francke,et al.  Small evolutionarily conserved RNA, resembling C/D box small nucleolar RNA, is transcribed from PWCR1, a novel imprinted gene in the Prader-Willi deletion region, which Is highly expressed in brain. , 2000, American journal of human genetics.

[25]  D. Tollervey,et al.  Synthesis and Assembly of the Box C+D Small Nucleolar RNPs , 2000, Molecular and Cellular Biology.

[26]  B. Chait,et al.  ProFound: an expert system for protein identification using mass spectrometric peptide mapping information. , 2000, Analytical chemistry.

[27]  A. Hüttenhofer,et al.  Identification of brain-specific and imprinted small nucleolar RNA genes exhibiting an unusual genomic organization. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Christiane Branlant,et al.  A Common Core RNP Structure Shared between the Small Nucleoar Box C/D RNPs and the Spliceosomal U4 snRNP , 2000, Cell.

[29]  D. Kressler,et al.  Spb1p Is a Yeast Nucleolar Protein Associated with Nop1p and Nop58p That Is Able To BindS-Adenosyl-l-Methionine In Vitro , 2000, Molecular and Cellular Biology.

[30]  D. Boisvert,et al.  Crystal structure of a fibrillarin homologue from Methanococcus jannaschii, a hyperthermophile, at 1.6 Å resolution , 2000, The EMBO journal.

[31]  K. Hartmuth,et al.  Crystal structure of the spliceosomal 15.5kD protein bound to a U4 snRNA fragment. , 2000, Molecular cell.

[32]  E. Binet,et al.  The FtsJ/RrmJ Heat Shock Protein of Escherichia coli Is a 23 S Ribosomal RNA Methyltransferase* , 2000, The Journal of Biological Chemistry.

[33]  E. Fauman,et al.  RNA methylation under heat shock control. , 2000, Molecular cell.

[34]  T. Kiss,et al.  A small nucleolar guide RNA functions both in 2′‐O‐ribose methylation and pseudouridylation of the U5 spliceosomal RNA , 2001, The EMBO journal.

[35]  A. Omer,et al.  In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[36]  T. Kiss.  Small Nucleolar RNAs An Abundant Group of Noncoding RNAs with Diverse Cellular Functions , 2002, Cell.