The spacing between functional Cis-elements of U3 snoRNA is critical for rRNA processing.

The sequences and structural features of Xenopus laevis U3 small nucleolar RNA (snoRNA) necessary for pre-rRNA cleavage at sites 1 and 2 to form 18 S rRNA were assayed by depletion/rescue experiments in Xenopus oocytes. Mutagenesis results demonstrated that the putative stem of U3 domain I is unnecessary for 18 S rRNA processing. A model consistent with earlier experimental data is proposed for the structure of domain I when U3 is not yet bound to pre-rRNA. For its function in rRNA processing, a newly discovered element (5' hinge) was revealed to be important but not as critical as the 3' hinge region in Xenopus U3 snoRNA for 18 S rRNA formation. Base-pairing is proposed to occur between the U3 5' hinge and 3' hinge and complementary regions in the external transcribed spacer (ETS); these interactions are phylogenetically conserved, and are homologous to those previously described in yeast (5' hinge-ETS) and trypanosomes (3' hinge-ETS). A model is presented where the base-pairing of the 5' hinge and 3' hinge of U3 snoRNA with the ETS of pre-rRNA helps to correctly position U3 boxes A'+A for their function in rRNA processing. Like an earlier proposal for yeast, boxes A' and A of Xenopus may base-pair with 18 S sequences in pre-rRNA. We present the first direct experimental evidence in any system that box A' is essential for U3 snoRNA function in 18 S rRNA formation. The analysis of insertions and deletions indicated that the spacing between the U3 elements is important, suggesting that they base-pair with the ETS and 18 S regions of pre-rRNA at the same time.

[1]  B. Maden,et al.  The external transcribed spacer and preceding region of Xenopus borealis rDNA: comparison with the corresponding region of Xenopus laevis rDNA. , 1983, Nucleic acids research.

[2]  R. Lührmann,et al.  An in vivo and in vitro structure-function analysis of the Saccharomyces cerevisiae U3A snoRNP: protein-RNA contacts and base-pair interaction with the pre-ribosomal RNA. , 1997, Journal of molecular biology.

[3]  J. Steitz,et al.  A new interaction between the mouse 5' external transcribed spacer of pre-rRNA and U3 snRNA detected by psoralen crosslinking. , 1992, Nucleic acids research.

[4]  D. Tollervey,et al.  Two distinct recognition signals define the site of endonucleolytic cleavage at the 5′‐end of yeast 18S rRNA. , 1995, The EMBO journal.

[5]  B. Sollner-Webb,et al.  Primary processing of mammalian rRNA involves two adjacent cleavages and is not species specific , 1987, Molecular and cellular biology.

[6]  D. Tollervey,et al.  Identification and functional analysis of two U3 binding sites on yeast pre‐ribosomal RNA. , 1992, The EMBO journal.

[7]  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.

[8]  S. Gerbi,et al.  Genes for Xenopus laevis U3 small nuclear RNA. , 1992, Nucleic acids research.

[9]  A. Weiner,et al.  Dictyostelium small nuclear RNA D2 is homologous to rat nucleolar RNA U3 and is encoded by a dispersed multigene family , 1980, Cell.

[10]  J. Bachellerie,et al.  Structure of the 5′‐external transcribed spacer of the human ribosomal RNA gene , 1989, FEBS letters.

[11]  R. Nazar,et al.  Essential structural features in the Schizosaccharomyces pombe pre-rRNA 5' external transcribed spacer. , 1999, Journal of molecular biology.

[12]  J. Steitz,et al.  The U3 small nucleolar ribonucleoprotein functions in the first step of preribosomal RNA processing , 1990, Cell.

[13]  J. Lee,et al.  Structure analysis of the 5' external transcribed spacer of the precursor ribosomal RNA from Saccharomyces cerevisiae. , 1990, Journal of molecular biology.

[14]  Véronique Ségault,et al.  An experimental study of Saccharomyces cerevisiae U3 snRNA conformation in solution , 1992, Nucleic Acids Res..

[15]  E. Maxwell,et al.  5'ETS rRNA processing facilitated by four small RNAs: U14, E3, U17, and U3. , 1996, RNA.

[16]  J. Steitz,et al.  U3, U8 and U13 comprise a new class of mammalian snRNPs localized in the cell nucleolus. , 1989, The EMBO journal.

[17]  M. O'Reilly,et al.  The terminal balls characteristic of eukaryotic rRNA transcription units in chromatin spreads are rRNA processing complexes. , 1993, Genes & development.

[18]  F. Solymosy,et al.  Molecular analysis of a U3 RNA gene locus in tomato: transcription signals, the coding region, expression in transgenic tobacco plants and tandemly repeated pseudogenes. , 1990, Nucleic acids research.

[19]  J. Bachellerie,et al.  Structure and organization of mouse U3B RNA functional genes. , 1988, The Journal of biological chemistry.

[20]  A. Weiner,et al.  The 5' end of U3 snRNA can be crosslinked in vivo to the external transcribed spacer of rat ribosomal RNA precursors. , 1989, Journal of molecular biology.

[21]  D. Tollervey,et al.  The 5' end of the 18S rRNA can be positioned from within the mature rRNA. , 1999, RNA.

[22]  J. Rientjes,et al.  Processing of eukaryotic pre-rRNA: the role of the transcribed spacers. , 1995, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[23]  W. Toyofuku,et al.  Two 5'-ETS regions implicated in interactions with U3 snoRNA are required for small subunit rRNA maturation in Trypanosoma brucei. , 1999, Nucleic acids research.

[24]  J. Steitz,et al.  A small nucleolar RNA is processed from an intron of the human gene encoding ribosomal protein S3. , 1993, Genes & development.

[25]  S. Gerbi,et al.  Nucleotide sequence determination and secondary structure of Xenopus U3 snRNA. , 1988, Nucleic acids research.

[26]  S. Baserga,et al.  Mpp10p, a U3 small nucleolar ribonucleoprotein component required for pre-18S rRNA processing in yeast , 1997, Molecular and cellular biology.

[27]  M. Ares,et al.  Evolution of eukaryotic rRNA: constraints imposed by RNA interactions. , 1987, Cold Spring Harbor symposia on quantitative biology.

[28]  S. Gerbi,et al.  U3 small nucleolar RNA is essential for cleavage at sites 1, 2 and 3 in pre-rRNA and determines which rRNA processing pathway is taken in Xenopus oocytes. , 1999, Journal of molecular biology.

[29]  D. Tollervey,et al.  The small nucleolar RNP protein NOP1 (fibrillarin) is required for pre‐rRNA processing in yeast. , 1991, The EMBO journal.

[30]  S. Gerbi,et al.  Nucleolar localization elements of Xenopus laevis U3 small nucleolar RNA. , 1998, Molecular biology of the cell.

[31]  R. Nazar,et al.  The ribosomal-RNA-processing pathway in Schizosaccharomyces pombe. , 1997, European journal of biochemistry.

[32]  J. Hughes,et al.  Functional base-pairing interaction between highly conserved elements of U3 small nucleolar RNA and the small ribosomal subunit RNA. , 1996, Journal of molecular biology.

[33]  J. Steitz,et al.  Structural analysis of the human U3 ribonucleoprotein particle reveal a conserved sequence available for base pairing with pre-rRNA. , 1987, Molecular and cellular biology.

[34]  F. Brulé,et al.  Secondary structure of the yeast Saccharomyces cerevisiae pre-U3A snoRNA and its implication for splicing efficiency. , 1996, RNA.

[35]  David Tollervey,et al.  Base Pairing between U3 Small Nucleolar RNA and the 5′ End of 18S rRNA Is Required for Pre-rRNA Processing , 1999, Molecular and Cellular Biology.

[36]  J. Wise,et al.  The sequence of U3 from Schizosaccharomyces pombe suggests structural divergence of this snRNA between metazoans and unicellular eukaryotes. , 1988, Nucleic acids research.

[37]  G. Cesareni,et al.  The yeast homologue of U3 snRNA. , 1987, The EMBO journal.

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

[39]  J. Wise,et al.  The two similarly expressed genes encoding U3 snRNA in Schizosaccharomyces pombe lack introns. , 1992, Molecular biology and evolution.

[40]  C. Branlant,et al.  An intron in the genes for U3 small nucleolar RNAs of the yeast Saccharomyces cerevisiae. , 1990, Science.

[41]  J. Bachellerie,et al.  Secondary structure of the 5' external transcribed spacer of vertebrate pre-rRNA. Presence of phylogenetically conserved features. , 1991, European journal of biochemistry.

[42]  M. Fournier,et al.  Functional Mapping of the U3 Small Nucleolar RNA from the Yeast Saccharomyces cerevisiae , 1998, Molecular and Cellular Biology.

[43]  M. Ares,et al.  Depletion of U3 small nucleolar RNA inhibits cleavage in the 5′ external transcribed spacer of yeast pre‐ribosomal RNA and impairs formation of 18S ribosomal RNA. , 1991, The EMBO journal.

[44]  D. Tollervey,et al.  The yeast Hansenula wingei U3 snoRNA gene contains an intron and its coding sequence co-evolved with the 5' ETS region of the pre-ribosomal RNA. , 1996, RNA: A publication of the RNA Society.

[45]  J. Bachellerie,et al.  Sequence and secondary structure of the 5' external transcribed spacer of mouse pre-rRNA. , 1988, DNA.

[46]  D. Tollervey,et al.  A U3 snoRNP protein with homology to splicing factor PRP4 and G beta domains is required for ribosomal RNA processing. , 1993, The EMBO journal.

[47]  R. Terns,et al.  Role of the box C/D motif in localization of small nucleolar RNAs to coiled bodies and nucleoli. , 1999, Molecular biology of the cell.

[48]  H. Nielsen,et al.  Sequence and proposed secondary structure of the Tetrahymena thermophila U3-snRNA. , 1993, Nucleic acids research.

[49]  T. Hartshorne.  Distinct regions of U3 snoRNA interact at two sites within the 5' external transcribed spacer of pre-rRNAs in Trypanosoma brucei cells. , 1998, Nucleic acids research.

[50]  S. Gerbi,et al.  In vivo disruption of Xenopus U3 snRNA affects ribosomal RNA processing. , 1990, The EMBO journal.

[51]  T. Hartshorne,et al.  A common core structure for U3 small nucleolar RNAs. , 1994, Nucleic acids research.

[52]  W. Filipowicz,et al.  Amplification of plant U3 and U6 snRNA gene sequences using primers specific for an upstream promoter element and conserved intragenic regions. , 1990, Nucleic acids research.

[53]  S. Gerbi.  Evolution of Ribosomal DNA , 1985 .

[54]  H. Busch,et al.  Multiple states of U3 RNA in Novikoff hepatoma nucleoli. , 1984, Biochemistry.

[55]  D. Tollervey,et al.  Base pairing between U3 and the pre‐ribosomal RNA is required for 18S rRNA synthesis. , 1995, The EMBO journal.