Borovjagin AV , Gerbi SA .

GM61945 NIGMS NIH HHS , R01 GM061945 NIGMS NIH HHS

	Figure 1. Cleavage sites in rRNA processing. Upper panel: comparison of the U3-dependent cleavage sites in yeast and Xenopus, which share in common site A0, site A1/1 and perhaps site A2/3. U3-dependent cleavage at sites A′ and 2 is found in metazoa but not in yeast. Lower panel: Xenopus pre-rRNA processing pathways. Pathways A and B differ in whether site 3 or sites A0, 1 and 2 are cleaved first. Pathways A and B can co-exist in a single cell, but some frogs just use pathway A (6). Note that the 20S intermediate is diagnostic for pathway A and the 36S intermediate is diagnostic for pathway B. Conversion of 20S to 18S occurs rapidly and the transient 19S and 18.5S intermediates are not always seen.
	Figure 2. U3 snoRNA interaction with the ETS. Base pairing between the 3′-hinge (3′H) of U3 snoRNA and region E1 of the pre-rRNA ETS is critical and sufficient for rRNA processing in Xenopus, whereas base pairing between the U3 5′-hinge (5′H) and region E2 of the ETS is auxiliary (26). In contrast, the 5′H–E2 interaction is essential in yeast and the 3′H–E1 interaction is not sufficient to support rRNA processing (27). Domain II of U3 snoRNA has many base-paired stems and binds several proteins (indicated by shading). Domain I of U3 is shown in an open configuration that can interact with the 5′ end of the 18S region of pre-rRNA (9,26,28). The conserved sequences comprising boxes that are conserved in U3 snoRNA from all species are indicated. Base pairing of U3 box A with the first terminal loop in 18S rRNA is thought to prevent premature pseudoknot formation (29,30). The pre-rRNA cleavage sites A0 and 1 are close to one another in the proposed secondary structure of the ETS (9,31).
	Figure 3. Base-pairing disruption of the U3 3′-hinge with ETS region E1. Intact endogenous U3 snoRNA was depleted by antisense oligonucleotide injection into oocyte nuclei. Subsequently, synthetic wild-type or mutated U3 was injected, and the restoration of 18S rRNA production was assayed by in vivo labeling with 32P-UTP, gel electrophoresis and autoradiography. The amount of 18S rRNA produced was quantified (see Materials and Methods) to normalize for any loading differences between gel lanes. Left panel: increasing disruption of base pairing at the left side of the 3′H–E1 interaction; right panel: increasing disruption of base pairing at the right side of the 3′H–E1 interaction.
	Figure 4. Restoration of base pairing of the U3 3′-hinge with ETS region E1. A plasmid with tagged pre-rRNA carrying mutations in ETS regions E1 and E2 was co-injected into Xenopus oocyte nuclei with an increasing number of substituted bases in the 3′-hinge (3′H) of U3 snoRNA. The number of base pairs between the U3 3′H and ETS region E1 required for the production of 18S rRNA was analyzed by Northern blots using a probe against the sequence tag in the 18S coding region of the injected rRNA plasmid. Less 40S pre-rRNA is seen in lanes 3 and 9 where there was complete base pairing between the U3 hinge and the ETS than in the mutations that shortened the base-pair interaction, suggesting that 40S pre-rRNA was processed more rapidly in the wild-type situation.
	Figure 5. 18S rRNA production requires precise positioning of base pairing between the U3 hinge and the ETS. U3 snoRNA containing sequence substitutions in the hinges or flanking regions was co-injected with a plasmid containing tagged 18S rRNA and substitutions in ETS E1 or E2 (see Table 1 for the sequences used). The mutated U3 hinges and mutated ETS regions E1 and E2 are indicated by open boxes, whereas the wild-type sequence for these areas is shown by a blackened box. Domain I and domain II of U3 snoRNA is marked. Shifts in the relative position of U3 relative to the pre-rRNA are denoted by an arrow. The production of tagged 18S rRNA after these treatments was assayed by Northern blots using a probe complementary to the 18S tag sequence. Internal controls are used for each panel because the rate of 40S pre-rRNA processing can vary between frogs (e.g. compare lanes 5 and 13).
	Figure 6. A compensatory shift in base pairing between U3 snoRNA and the ETS restores the relative position of U3 on pre-rRNA and 18S rRNA can be produced. After antisense oligonucleotide-mediated depletion of endogenous U3 snoRNA, Xenopus oocyte nuclei were injected with 1× or 3× amounts of synthetic transcripts of U3 carrying a sequence substitution in the 3′-hinge or the region immediately upstream to pair with the ETS of endogenous wild-type pre-rRNA. Open boxes show regions of sequence substitution, and blackened boxes show regions of wild-type sequence for the U3 hinges and for ETS region E1 and E2. The production of 18S rRNA was assayed by gel electrophoresis and autoradiography of 32P-UTP in vivo labeled RNA.

Beltrame, Base pairing between U3 and the pre-ribosomal RNA is required for 18S rRNA synthesis. 1995, Pubmed

Beltrame, Base pairing between U3 and the pre-ribosomal RNA is required for 18S rRNA synthesis. 1995, Pubmed
Bernstein, The small-subunit processome is a ribosome assembly intermediate. 2004, Pubmed
Borovjagin, 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, Pubmed , Xenbase
Borovjagin, Xenopus U3 snoRNA GAC-Box A' and Box A sequences play distinct functional roles in rRNA processing. 2001, Pubmed , Xenbase
Borovjagin, The spacing between functional Cis-elements of U3 snoRNA is critical for rRNA processing. 2000, Pubmed , Xenbase
Borovjagin, Xenopus U3 snoRNA docks on pre-rRNA through a novel base-pairing interaction. 2004, Pubmed , Xenbase
Dragon, A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis. 2002, Pubmed
Fatica, Nob1p is required for cleavage of the 3' end of 18S rRNA. 2003, Pubmed
Fatica, PIN domain of Nob1p is required for D-site cleavage in 20S pre-rRNA. 2004, Pubmed
Gallagher, RNA polymerase I transcription and pre-rRNA processing are linked by specific SSU processome components. 2004, Pubmed
Gao, Human genes encoding U3 snRNA associate with coiled bodies in interphase cells and are clustered on chromosome 17p11.2 in a complex inverted repeat structure. 1997, Pubmed
Gerbi, Ribosome biogenesis: role of small nucleolar RNA in maturation of eukaryotic rRNA. 2001, Pubmed
Ginisty, Nucleolin functions in the first step of ribosomal RNA processing. 1998, Pubmed , Xenbase
Ginisty, Two different combinations of RNA-binding domains determine the RNA binding specificity of nucleolin. 2001, Pubmed , Xenbase
Grandi, 90S pre-ribosomes include the 35S pre-rRNA, the U3 snoRNP, and 40S subunit processing factors but predominantly lack 60S synthesis factors. 2002, Pubmed
Granneman, The human Imp3 and Imp4 proteins form a ternary complex with hMpp10, which only interacts with the U3 snoRNA in 60-80S ribonucleoprotein complexes. 2003, Pubmed
Granneman, Role of pre-rRNA base pairing and 80S complex formation in subnucleolar localization of the U3 snoRNP. 2004, Pubmed
Gérczei, Imp3p and Imp4p mediate formation of essential U3-precursor rRNA (pre-rRNA) duplexes, possibly to recruit the small subunit processome to the pre-rRNA. 2004, Pubmed
Hughes, Functional base-pairing interaction between highly conserved elements of U3 small nucleolar RNA and the small ribosomal subunit RNA. 1996, Pubmed
Hughes, 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, Pubmed
Intine, Essential structural features in the Schizosaccharomyces pombe pre-rRNA 5' external transcribed spacer. 1999, Pubmed
Lange, Nucleolar localization elements of Xenopus laevis U3 small nucleolar RNA. 1998, Pubmed , Xenbase
Lee, Imp3p and Imp4p, two specific components of the U3 small nucleolar ribonucleoprotein that are essential for pre-18S rRNA processing. 1999, Pubmed
Mougey, The terminal balls characteristic of eukaryotic rRNA transcription units in chromatin spreads are rRNA processing complexes. 1993, Pubmed , Xenbase
Méreau, 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, Pubmed
Narayanan, Role of the box C/D motif in localization of small nucleolar RNAs to coiled bodies and nucleoli. 1999, Pubmed , Xenbase
Osheim, Pre-18S ribosomal RNA is structurally compacted into the SSU processome prior to being cleaved from nascent transcripts in Saccharomyces cerevisiae. 2004, Pubmed
Peculis, snoRNA nuclear import and potential for cotranscriptional function in pre-rRNA processing. 2001, Pubmed , Xenbase
Savino, In vivo disruption of Xenopus U3 snRNA affects ribosomal RNA processing. 1990, Pubmed , Xenbase
Schäfer, The path from nucleolar 90S to cytoplasmic 40S pre-ribosomes. 2003, Pubmed
Sharma, The 5' end of the 18S rRNA can be positioned from within the mature rRNA. 1999, Pubmed
Sharma, Base pairing between U3 small nucleolar RNA and the 5' end of 18S rRNA is required for pre-rRNA processing. 1999, Pubmed
Tabb-Massey, Ribosomal proteins Rps0 and Rps21 of Saccharomyces cerevisiae have overlapping functions in the maturation of the 3' end of 18S rRNA. 2003, Pubmed
Vanrobays, Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein. 2001, Pubmed
Vanrobays, Late cytoplasmic maturation of the small ribosomal subunit requires RIO proteins in Saccharomyces cerevisiae. 2003, Pubmed
Venema, Ribosome synthesis in Saccharomyces cerevisiae. 1999, Pubmed
Venema, Two distinct recognition signals define the site of endonucleolytic cleavage at the 5'-end of yeast 18S rRNA. 1995, Pubmed
Verheggen, Box C/D small nucleolar RNA trafficking involves small nucleolar RNP proteins, nucleolar factors and a novel nuclear domain. 2001, Pubmed
Verheggen, Mammalian and yeast U3 snoRNPs are matured in specific and related nuclear compartments. 2002, Pubmed , Xenbase
Warner, The economics of ribosome biosynthesis in yeast. 1999, Pubmed
Zuker, Mfold web server for nucleic acid folding and hybridization prediction. 2003, Pubmed