The Small Nucle(ol)ar RNA Cap Trimethyltransferase Is Required for Ribosome Synthesis and Intact Nucleolar Morphology

ABSTRACT Nucleolar morphogenesis is a poorly defined process. Here we report that the Saccharomyces cerevisiae nucleolar trimethyl guanosine synthase I (Tgs1p), which specifically selects the m7G cap structure of snRNAs and snoRNAs for m2,2,7G conversion, is required not only for efficient pre-mRNA splicing but also for pre-rRNA processing and small ribosomal subunit synthesis. Mutational analysis indicates that the requirement for Tgs1p in pre-mRNA splicing, but not its involvement in ribosome synthesis, is dependent upon its function in cap trimethylation. In addition, we report that cells lacking Tgs1p showed a striking and unexpected loss of nucleolar structural organization. Tgs1p is not a core component of the snoRNP proteins; however, in vitro, the protein interacts with the KKD/E domain present at the carboxyl-terminal ends of several snoRNP proteins. Strains expressing versions of the snoRNPs lacking the KKD/E domain were also defective for nucleolar morphology and showed a loss of nucleolar compaction. We propose that the transient and functional interactions of Tgs1p with the abundant snoRNPs, through presumed interactions with the KKD/E domain of the snoRNP proteins, contribute substantially to the coalescence of nucleolar components. This conclusion is compatible with a model of self-organization for nucleolar assembly.

[1]  M. Okuwaki,et al.  [Structure and function of the nucleolus]. , 2006, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[2]  D. Lafontaine,et al.  Dim2p, a KH-domain protein required for small ribosomal subunit synthesis. , 2004, RNA.

[3]  J. Bujnicki,et al.  Sequence-structure-function relationships of Tgs1, the yeast snRNA/snoRNA cap hypermethylase. , 2003, Nucleic acids research.

[4]  E. Petfalski,et al.  The path from nucleolar 90S to cytoplasmic 40S pre‐ribosomes , 2003, The EMBO journal.

[5]  P. Thuriaux,et al.  Hmo1, an HMG‐box protein, belongs to the yeast ribosomal DNA transcription system , 2002, The EMBO journal.

[6]  E. Petfalski,et al.  Rlp7p is associated with 60S preribosomes, restricted to the granular component of the nucleolus, and required for pre-rRNA processing , 2002, The Journal of cell biology.

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

[8]  J. Tazi,et al.  Hypermethylation of the cap structure of both yeast snRNAs and snoRNAs requires a conserved methyltransferase that is localized to the nucleolus. , 2002, Molecular cell.

[9]  S. Kuersten,et al.  Nucleocytoplasmic transport: Ran, beta and beyond. , 2001, Trends in cell biology.

[10]  T. Misteli.  The concept of self-organization in cellular architecture , 2001, The Journal of cell biology.

[11]  D. Tollervey,et al.  Box C/D small nucleolar RNA trafficking involves small nucleolar RNP proteins, nucleolar factors and a novel nuclear domain , 2001, The EMBO journal.

[12]  C. Will,et al.  Spliceosomal UsnRNP biogenesis, structure and function. , 2001, Current opinion in cell biology.

[13]  Sui Huang,et al.  Nucleolar Components Involved in Ribosome Biogenesis Cycle between the Nucleolus and Nucleoplasm in Interphase Cells , 2001, The Journal of cell biology.

[14]  T Misteli,et al.  Protein dynamics: implications for nuclear architecture and gene expression. , 2001, Science.

[15]  M. Dundr,et al.  The nucleolus: an old factory with unexpected capabilities. , 2000, Trends in cell biology.

[16]  T. Misteli,et al.  High mobility of proteins in the mammalian cell nucleus , 2000, Nature.

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

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

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

[20]  M. Mann,et al.  Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure. , 1998, RNA.

[21]  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.

[22]  D. Tollervey,et al.  Yeast 18S rRNA Dimethylase Dim1p: a Quality Control Mechanism in Ribosome Synthesis? , 1998, Molecular and Cellular Biology.

[23]  D. Tollervey,et al.  The box H + ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase. , 1998, Genes & development.

[24]  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.

[25]  D. Tollervey,et al.  One-step PCR mediated strategy for the construction of conditionally expressed and epitope tagged yeast proteins. , 1996, Nucleic acids research.

[26]  J. Vandenhaute,et al.  The 18S rRNA dimethylase Dim1p is required for pre-ribosomal RNA processing in yeast. , 1995, Genes & development.

[27]  Leon D. Segal,et al.  Functions , 1995 .

[28]  F. Larimer,et al.  Fragments of the internal transcribed spacer 1 of pre-rRNA accumulate in Saccharomyces cerevisiae lacking 5'----3' exoribonuclease 1 , 1991, Journal of bacteriology.

[29]  D. Lafontaine,et al.  Eukaryotic Ribosome Synthesis , 2004 .

[30]  David Tollervey,et al.  Making ribosomes. , 2002, Current opinion in cell biology.

[31]  M. Thiry.  Ultrastructural methods for nucleic acid detection by immunocytology. , 1999, Progress in histochemistry and cytochemistry.

[32]  D. Tollervey,et al.  Ribosome synthesis in Saccharomyces cerevisiae. , 1999, Annual review of genetics.

[33]  I. Mattaj,et al.  Nucleocytoplasmic transport: the soluble phase. , 1998, Annual review of biochemistry.

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

[35]  B. Haarer,et al.  Immunofluorescence methods for yeast. , 1991, Methods in enzymology.

[36]  E. Weibel.  Stereological principles for morphometry in electron microscopic cytology. , 1969, International review of cytology.