Identification and functional analysis of two U3 binding sites on yeast pre-ribosomal RNA.

It has long been known that U3 can be isolated hydrogen bonded to pre-ribosomal RNAs, but the sites of interaction are poorly characterized. Here we show that yeast U3 can be cross-linked to 35S pre-rRNA both in deproteinized extracts and in living cells. The sites of cross-linking were localized to the 5' external transcribed spacer (ETS) and then identified at the nucleotide level. Two regions of U3 near the 5' end are cross-linked to pre-rRNA in vivo and in vitro; the evolutionarily conserved box A region and a 10 nucleotide (nt) sequence with perfect complementarity to an ETS sequence. Two in vivo cross-links are detected in the ETS, at +470, within the region complementary to U3, and at +655, close to the cleavage site at the 5' end of 18S rRNA. A tagged rDNA construct was used to follow the effects of mutations in the ETS in vivo. A small deletion around the +470 cross-linking site in the ETS prevents the synthesis of 18S rRNA. This region is homologous to the site of vertebrate ETS cleavage. We propose that this site may be evolutionarily conserved to direct the assembly of a pre-rRNA processing complex required for the cleavages that generate 18S rRNA.     

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.

The 5' external transcribed spacer (ETS) region of the pre-rRNA in Saccharomyces cerevisiae contains a sequence with 10 bp of perfect complementarity to the U3 snoRNA. Base pairing between these sequences has been shown to be required for 18S rRNA synthesis, although interaction over the full 10 bp of complementarity is not required. We have identified the homologous sequence in the 5' ETS from the evolutionarily distant yeast Hansenula wingei; unexpectedly, this shows two sequence changes in the region predicted to base pair to U3. By PCR amplification and direct RNA sequencing, a single type of U3 snoRNA coding sequence was identified in H. wingei. As in the S. cerevisiae U3 snoRNA genes, it is interrupted by an intron with features characteristic of introns spliced in a spliceosome. Consequently, this unusual property is not restricted to the yeast genus Saccharomyces. The introns of the H. wingei and S. cerevisiae U3 genes show strong differences in length and sequence, but are located at the same position in the U3 sequence, immediately upstream of the phylogenetically conserved Box A region. The 3' domains of the H. wingei and S. cerevisiae U3 snoRNAs diverge strongly in primary sequence, but have very similar predicted secondary structures. The 5' domains, expected to play a direct role in pre-ribosomal RNA maturation, are more conserved. The sequence predicted to base pair to the pre-rRNA contains two nucleotide substitutions in H. wingei that restore 10 bp of perfect complementarity to the 5' ETS. This is a strong phylogenetic evidence for the importance of the U3/pre-rRNA interaction.     

Structural and functional analysis of the U3 snoRNA binding protein Rrp9

The U3 snoRNA is required for 18S rRNA processing and small subunit ribosome formation in eukaryotes. Different from other box C/D snoRNAs, U3 contains an extra 5′ domain that pairs with pre-rRNA and a unique B/C motif essential for recruitment of the U3-specific Rrp9 protein. Here, we analyze the structure and function of Rrp9 with crystallographic, biochemical, and cellular approaches. Rrp9 is composed of a WD repeat domain and an N-terminal region. The crystal structures of the WD domain of yeast Rrp9 and its human ortholog U3-55K were determined, revealing a typical seven-bladed propeller fold. Several conserved surface patches on the WD domain were identified, and their function in RNP assembly and yeast growth were analyzed by mutagenesis. Prior association of Snu13 with the B/C motif was found to enhance the specific binding of the WD domain. We show that a conserved 7bc loop is crucial for specific recognition of U3, nucleolar localization of Rrp9, and yeast growth. In addition, we show that the N-terminal region of Rrp9 contains a bipartite nuclear localization signal that is dispensable for nucleolar localization. Our results provide insight into the functional sites of Rrp9.     

Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.

The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.     

Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.

The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.     

A weak C' box renders U3 snoRNA levels dependent on hU3-55K binding

The rate of ribosome biogenesis, which is downregulated in terminally differentiated cells and upregulated in most cancers, regulates the growth rate and is linked to the cell's proliferative potential. The U3 box C/D small nucleolar RNP (snoRNP) is an integral component of the small subunit (SSU) processome and is essential for 18S rRNA processing. We show that U3 snoRNP assembly, and therefore U3 snoRNA accumulation, is regulated through the U3-specific protein hU3-55K. Furthermore, we report that the levels of several SSU processome components, including the U3 snoRNA but not other box C/D snoRNAs, are specifically downregulated during human lung (CaCo-2) and colon (CaLu-3) epithelial cell differentiation. c-Myc is reported to play an integral role in regulating ribosome production by controlling the expression of many ribosome biogenesis factors. Our data, however, indicate that this regulation is not dependent on c-Myc since the level of this protein does not change during epithelial cell differentiation. In addition, depletion of c-Myc had only a mild affect on the levels of SSU processome proteins. CaCo-2 cells are colon adenocarcinoma epithelial cells that are believed to revert to their precancerous state during differentiation. This suggests a significant increase in the levels of specific SSU processome components during tumorogenesis.     

An axial binding site in the Tetrahymena precursor RNA.

Previous studies allow the construction of three distinct models of the binding of G and arginine within the active site of the Tetrahymena self-splicing preribosomal precursor RNA. These models (base triple, axial I and axial II) are now distinguished by measurements on the specificity of RNAs with nucleotide substitutions at positions spanning the site. Because the semi-conserved unpaired nucleotide 263 has no effect on substrate or inhibitor selection by the Tetrahymena RNA we conclude that the axial I model is improbable. In contrast, data with substituted RNAs and nucleoside analogs suggest that nucleotide 265 makes a hydrogen bond with the substrate. Accordingly the active site appears axial because substrate contacts exist at more than one nucleotide on the 5' side of the P7 helix. The effects of this hydrogen bond are observable in cases where the donor or acceptor is on the RNA, whether nucleotide 265 is a purine or pyrimidine, or whether nucleotide 265 is mispaired, wobble paired or normally paired. This pattern is consistent with the axial II model. Molecular dynamics and energy minimization calculations lead to the same conclusions as these site-directed substitutions; the base triple and axial I models are unstable dynamically. Under thermal agitation, the third model site (axial II) is transformed to a related, but more stable structure, axial III. The axial III active site is characterized by the extrusion of the conserved bulged base 263 from the P7 helix, a semi-pocket for G base formed by stacking of nucleotide 262, and formation of all bonds to the G base originally proposed for both the base triple and axial II sites. Because of these hydrogen bonds the axial III site is also consistent with data on enzymatic specificity. The axial III model indicates an unforeseen capacity for pocket formation within the groove of an RNA helix, suggests that the site may be unusually flexible, and bears on a hypothesis concerning the origin of the genetic code.     

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

The nucleolus, the site of pre-ribosomal RNA (pre-rRNA) synthesis and processing in eukaryotic cells, contains a number of small nucleolar RNAs (snoRNAs). Yeast U3 snoRNA is required for the processing of 18S rRNA from larger precursors and contains a region complementary to the pre-rRNA. Substitution mutations in the pre-rRNA which disrupt this base pairing potential are lethal and prevent synthesis of 18S rRNA. These mutant pre-rRNAs show defects in processing which closely resemble the effects of genetic depletion of components of the U3 snoRNP. Co-expression of U3 snoRNAs which carry compensatory mutations allows the mutant pre-rRNAs to support viability and synthesize 18S rRNA at high levels. Pre-rRNA processing steps which are blocked by the external transcribed spacer region mutations are largely restored by expression of the compensatory U3 mutants. Pre-rRNA processing therefore requires direct base pairing between snoRNA and the substrate. Base pairing with the substrate is thus a common feature of small RNAs involved in mRNA and rRNA maturation.     

U3 snoRNA genes with and without intron in the Kluyveromyces genus: yeasts can accommodate great variations of the U3 snoRNA 3'-terminal domain.

The U3 snoRNA coding sequences from the genomic DNAs of Kluyveromyces delphensis and four variants of the Kluyveromyces marxianus species were cloned by PCR amplification. Nucleotide sequence analysis of the amplification products revealed a unique U3 snoRNA gene sequence in all the strains studied, except for K. marxianus var. fragilis. The K. marxianus U3 genes were intronless, whereas an intron similar to those of the Saccharomyces cerevisiae U3 genes was found in K. delphensis. Hence, U3 genes with and without intron are found in yeasts of the Saccharomycetoideae subfamily. The secondary structure of the K. delphensis pre-U3 snoRNA and of the K. marxianus mature snoRNAs were studied experimentally. They revealed a strong conservation in yeasts of (1) the architecture of U3 snoRNA introns, (2) the 5'-terminal domain of the mature snoRNA, and (3) the protein-anchoring regions of the U3 snoRNA 3' domain. In contrast, stem-loop structures 2, 3, and 4 of the 3' domain showed great variations in size, sequence, and structure. Using a genetic test, we show that, in spite of these variations, the Kluyveromyces U3 snoRNAs are functional in S. cerevisiae. We also show that S. cerevisiae U3A snoRNAs lacking the stem-loop structure 2 or 4 are functional. Hence, U3 snoRNA function can accommodate great variations of the RNA 3'-terminal domain.     

Utp14 Recruits and Activates the RNA Helicase Dhr1 To Undock U3 snoRNA from the Preribosome

In eukaryotic ribosome biogenesis, U3 snoRNA base pairs with the pre-rRNA to promote its processing. However, U3 must be removed to allow folding of the central pseudoknot, a key feature of the small subunit. Previously, we showed that the DEAH/RHA RNA helicase Dhr1 dislodges U3 from the pre-rRNA. DHR1 can be linked to UTP14, encoding an essential protein of the preribosome, through genetic interactions with the rRNA methyltransferase Bud23. Here, we report that Utp14 regulates Dhr1. Mutations within a discrete region of Utp14 reduced interaction with Dhr1 that correlated with reduced function of Utp14. These mutants accumulated Dhr1 and U3 in a pre-40S particle, mimicking a helicase-inactive Dhr1 mutant. This similarity in the phenotypes led us to propose that Utp14 activates Dhr1. Indeed, Utp14 formed a complex with Dhr1 and stimulated its unwinding activity in vitro. Moreover, the utp14 mutants that mimicked a catalytically inactive dhr1 mutant in vivo showed reduced stimulation of unwinding activity in vitro. Dhr1 binding to the preribosome was substantially reduced only when both Utp14 and Bud23 were depleted. Thus, Utp14 is bifunctional; together with Bud23, it is needed for stable interaction of Dhr1 with the preribosome, and Utp14 activates Dhr1 to dislodge U3.     

Identification of the RNA binding segment of human U1 A protein and definition of its binding site on U1 snRNA.

The interaction between the U1 snRNP-specific U1 A protein and U1 snRNA has been analysed. The binding site for the protein on the RNA is shown to be in hairpin II, which extends from positions 48 to 91 in the RNA. Within this hairpin the evolutionarily conserved loop sequence is crucial for interaction with U1 A protein. U1 A protein can also bind the loop sequence when it is part of an artificial RNA which cannot form a stable hairpin structure. The region of the protein required to bind to U1 snRNA consists of a conserved 80 amino acid motif, previously identified in many ribonucleoprotein (RNP) proteins, together with (maximally) 11 N-terminal and 10 C-terminal flanking amino acids. Point mutations introduced into two of the most highly conserved regions of this motif abolish RNA binding. U1 snRNA mutants from which the U1 A binding site has been deleted are shown to be capable of assembly into RNP particles which are immunoprecipitable by patient antisera which recognize U1 A protein. The role of RNA-protein and protein-protein interactions in U snRNP assembly are discussed.     

U3 snoRNA may recycle through different compartments of the nucleolus

A model is proposed in which U3 small nucleolar RNA (snoRNA) is recruited from an inactive, stored form in the dense fibrillar component (DFC) of the nucleolus to an active form that is associated with the initial ribosomal RNA (rRNA) precursor. The initial steps of rRNA processing occur in the DFC, and then it is proposed that the U3 snoRNA moves with intermediates in rRNA processing from the DFC to the granular component (GC) of the nucleolus. The nucleolar protein fibrillarin is located primarily in the DFC, and it is suggested that the complex of fibrillarin and U3 snoRNA dissociates when U3 snoRNA transits to the GC. Finally, when U3 snoRNA is released from the processed rRNA, the tether holding the rRNA in the nucleolus is broken and rRNA can then be exported from the nucleolus to the cytoplasm. U3 snoRNA is hypothesized to recycle back from the GC to the DFC where it is stored until future association with another initial rRNA precursor. Data supporting this model are summarized. U3 snoRNA is also stored in the coiled body of interphase cells and in the nucleolar remnants and prenucleolar bodies of mitotic cells, and there may be some similarity in the binding sites for stored U3 snoRNA in the DFC and in these structures. Received: 16 September 1996 / Accepted: 11 November 1996     

A complex pathway for 3' processing of the yeast U3 snoRNA

Mature U3 snoRNA in yeast is generated from the 3′-extended precursors by endonucleolytic cleavage followed by exonucleolytic trimming. These precursors terminate in poly(U) tracts and are normally stabilised by binding of the yeast La homologue, Lhp1p. We report that normal 3′ processing of U3 requires the nuclear Lsm proteins. On depletion of any of the five essential proteins, Lsm2–5p or Lsm8p, the normal 3′-extended precursors to the U3 snoRNA were lost. Truncated fragments of both mature and pre-U3 accumulated in the Lsm-depleted strains, consistent with substantial RNA degradation. Pre-U3 species were co-precipitated with TAP-tagged Lsm3p, but the association with spliced pre-U3 was lost in strains lacking Lhp1p. The association of Lhp1p with pre-U3 was also reduced on depletion of Lsm3p or Lsm5p, indicating that binding of Lhp1p and the Lsm proteins is interdependent. In contrast, a tagged Sm-protein detectably co-precipitated spliced pre-U3 species only in strains lacking Lhp1p. We propose that the Lsm2–8p complex functions as a chaperone in conjunction with Lhp1p to stabilise pre-U3 RNA species during 3′ processing. The Sm complex may function as a back-up to stabilise 3′ ends that are not protected by Lhp1p.     

Xenopus U3 snoRNA docks on pre-rRNA through a novel base-pairing interaction

U3 small nucleolar RNA (snoRNA) is essential for rRNA processing to form 18S ribosomal RNA (rRNA). Previously, it has been shown that nucleolin is needed to load U3 snoRNA on pre-rRNA. However, as documented here, this is not sufficient. We present data that base-pairing between the U3 hinges and the external transcribed spacer (ETS) is critical for functional alignment of U3 on its pre-rRNA substrate. Additionally, the interaction between the U3 hinges and the ETS is proposed to serve as an anchor to hold U3 on the pre-rRNA substrate, while box A at the 5' end of U3 snoRNA swivels from ETS contacts to 18S rRNA contacts. Compensatory base changes revealed base-pairing between the 3' hinge of U3 snoRNA and region E1 of the ETS in Xenopus pre-rRNA; this novel interaction is required for 18S rRNA production. In contrast, base-pairing between the 5' hinge of U3 snoRNA and region E2 of the ETS is auxiliary, unlike the case in yeast where it is required. Thus, higher and lower eukaryotes use different interactions for functional association of U3 with pre-rRNA. The U3 hinge sequence varies between species, but covariation in the ETS retains complementarity. This species-specific U3-pre-rRNA interaction offers a potential target for a new class of antibiotics to prevent ribosome biogenesis in eukaryotic pathogens.     

A second base pair interaction between U3 small nucleolar RNA and the 5'-ETS region is required for early cleavage of the yeast pre-ribosomal RNA

In eukaryotes, U3 snoRNA is essential for pre-rRNA maturation. Its 5'-domain was found to form base pair interactions with the 18S and 5'-ETS parts of the pre-rRNA. In Xenopus laevis, two segments of U3 snoRNA form base-pair interactions with the 5'-ETS region and only one of them is essential to the maturation process. In Saccharomyces cerevisiae, two similar U3 snoRNA-5' ETS interactions are possible; but, the functional importance of only one of them had been tested. Surprisingly, this interaction, which corresponds to the non-essential one in X. laevis, is essential for cell growth and pre-rRNA maturation in yeast. In parallel with [Dutca et al. (2011) The initial U3 snoRNA:pre-rRNA base pairing interaction required for pre-18S rRNA folding revealed by in vivo chemical probing. Nucleic Acids Research, 39, 5164-5180], here we show, that the second possible 11-bp long interaction between the 5' domain of S. cerevisiae U3 snoRNA and the pre-rRNA 5'-ETS region (helix VI) is also essential for pre-rRNA processing and cell growth. Compensatory mutations in one-half of helix VI fully restored cell growth. Only a partial restoration of growth was obtained upon extension of compensatory mutations to the entire helix VI, suggesting sequence requirement for binding of specific proteins. Accordingly, we got strong evidences for a role of segment VI in the association of proteins Mpp10, Imp4 and Imp3.     

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.

Multiple processing events are required to convert a single eukaryotic pre-ribosomal RNA (pre-rRNA) into mature 18S (small subunit), 5.8S and 25-28S (large subunit) rRNAs. We have asked whether U3 small nucleolar RNA is required for pre-rRNA processing in vivo by depleting Saccharomyces cerevisiae of U3 by conditional repression of U3 synthesis. The resulting pattern of accumulation and depletion of specific pre-rRNAs indicates that U3 is required for multiple events leading to the maturation of 18S rRNA. These include an initial cleavage within the 5' external transcribed spacer, resembling the U3 dependent initial processing event of mammalian pre-rRNA. Formation of large subunit rRNAs is unaffected by U3 depletion. The similarity between the effects of U3 depletion and depletion of U14 small nucleolar RNA and the nucleolar protein fibrillarin (NOP1) suggests that these could be components of a single highly conserved processing complex.