INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

The small nucleolar RNAs (snoRNAs) play essential roles in posttranscriptional maturation of rRNAs (reviewed in references 2, 17, 38, 40, 51, 65, and 69). A few snoRNAs are required for cleavage of rRNA precursors which encode the 18S, 5.8S, and 25S/28S rRNAs. Most and possibly all of the other snoRNAs serve as guide RNAs in nucleotide modification reactions, in particular ribose methylation and pseudouridine formation. Scores of snoRNAs have already been identified in protist, fungal, plant, and animal cells, and all are believed to exist as snoRNP particles. The various rRNA maturation reactions are thought to occur in a large, poorly defined complex called the processome (16; see also references 17 and 40).

U3, the subject of the present report, is one of a few phylogenetically conserved snoRNAs and appears to join the processome at an early, vital stage. In mammalian and yeast cells U3 is approximately 1 order of magnitude more abundant than other snoRNAs (58, 73). U3 is required for several early pre-rRNA cleavage reactions, including (i) an initial cleavage in the 5' external transcribed spacer (5' ETS) region (in yeast cells, mouse extracts, and Xenopus oocyte extracts [25, 29, 45]); (ii) two sites that flank the 18S rRNA coding region (in yeast cells [25]); and (iii) a site near the boundary of internal transcribed spacer-1 (ITS1) and 5.8S rRNA (Xenopus oocytes [60]). U3 appears to interact with pre-rRNA at a variety of sites. In yeast cells, U3 has been proposed to contact pre-rRNA at three sites: one in the 5' ETS portion and two early in the 18S rRNA coding region. The site in the 5' ETS was identified by in vivo cross-linking, and subsequent mutation analysis showed it to be essential for 18S rRNA production (7, 9). Additional support for this interaction came from a suppressor mutation in U3 (8; see also below). The two interactions within the 18S rRNA coding region are proposed to occur through base-pairing of a pseudoknot in U3 (24, 44). This suggestion is supported by strong conservation of the putative interacting sequences in both U3 and 18S RNA and by preliminary mutations in the corresponding U3 nucleotides which affect cell viability.

Progress in defining proteins associated with U3 is still at an early stage. For many years U3 RNAs have been known to be associated with the nucleolar protein fibrillarin, or its yeast homolog, Nop1p. This association, which is probably indirect, is now known to be characteristic of snoRNAs in one of two major snoRNA groups, i.e., the box C/D family; the second major family consists of box H/ACA snoRNAs (reviewed in references 65 and 69). Depletion of Nop1p from yeast cells causes severe defects in ribosome biogenesis, including cleavage and methylation of pre-rRNA and ribosome assembly (70, 71). More recently, other proteins associated with U3 have been identified. A U3 RNA-protein complex from CHO cells was shown to contain three previously uncharacterized proteins and seems likely to be a core particle of the native U3 snoRNP (37). Two proteins associated with yeast U3 have been identified, Sof1p and Mpp10p (15, 27). The gene for Sof1p was found in a search for suppressors of a temperature-sensitive growth phenotype caused by substituting the yeast NOP1 gene with its human homolog. The gene for Mpp10p was identified during a search for a structural homolog of a human protein (MPP10), which, in turn, had been detected with antibodies specific for proteins phosphorylated during mitosis. Neither yeast protein is known to be associated with other snoRNAs, as determined by limited hybridization screening of immunoprecipitated RNAs (Sof1p and Mpp10p) and analysis of immunoprecipitated and postlabeled RNAs (Mpp10p). It is not known if Sof1p and Mpp10p interact directly with U3. However, depletion of these proteins causes defects in rRNA processing similar to those resulting from loss or inactivation of U3 itself.

All known U3 RNAs contain a 5' cap, and the nature of the cap correlates with the type of RNA polymerase used in transcription. Protist, fungal, and animal U3 RNAs are transcribed by RNA polymerase II and have a trimethyl guanosine (TMG) cap. In plants U3 is produced by RNA polymerase III and has a gamma-monomethyl phosphate cap (reviewed in reference 17). In all cases, known U3 is transcribed from independent genes. This is in contrast to most other snoRNAs in animals, which are encoded within introns of protein genes, and some plant snoRNAs, which are transcribed from polycistronic snoRNA operons (reviewed in reference 69). The U3 genes in several species of fungi have the remarkable distinction of being the only snoRNA coding sequences known which themselves contain introns (11, 47).

Phylogenetic comparison of the first U3 snoRNAs revealed conserved sequence elements, called boxes A to D (26, 28, 30, 52, 73, 77). Additional elements, called boxes A' and C' (or A°), have been defined more recently (39, 47, 74). Other snoRNAs were subsequently determined to contain boxes C and D, and these elements now define the box C/D family of snoRNAs. Most box C/D snoRNAs possess long (>12 nucleotides) sequences complementary to rRNA that are located immediately upstream of box D and/or an analog box D' (reviewed in references 2, 65, and 69). RNAs with these features guide the formation of 2'-O-methylated nucleotides in rRNAs (14, 32, 48, 75). The U3 snoRNAs do not contain the methylation motif and thus are not believed to function in 2'-O-methylation.

Secondary structure models have been proposed for a variety of U3 molecules, based on computer folding, phylogenetic sequence comparisons, and direct structure probing data (10, 18, 21, 22, 26, 28, 30, 31, 34, 39, 41-44, 46, 47, 49, 50, 52, 53, 61, 62). A consensus structure has not yet been determined, but several features occur consistently (Fig. 1). The conserved box A' and A elements and adjoining nonconserved nucleotides, known as a hinge region, define the 5' portion of the molecule. No unambiguous secondary structure has been proposed for this region: one or two hairpins were proposed to be formed in yeasts, one or no hairpins were proposed for higher eukaryotes, and no hairpins were proposed for protists. The rest of the molecule is believed to be highly folded and relatively consistent structures have been proposed for this portion. Boxes C' and D occur between phylogenetically conserved terminal and central stems, juxtaposed next to each other. The regions surrounding boxes B and C are not conserved; however, in all cases boxes B and C are predicted to be in close proximity. Folding of the surrounding segments results in different numbers of hairpins: from none in protists to three in some fungal species.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   The U3 snoRNA from the yeast S. cerevisiae. The model shown is derived from a compilation of structures proposed for U3 RNAs from different organisms. Several configurations have been suggested for the 5' segment including (i) nonfolded (28, 49), (ii) possessing a single helix (hairpin 1 [50]), or (iii) containing two helices (hairpin 1a and 1b, not shown [44, 52]). The well-structured 3' portion of S. cerevisiae U3 contains two highly conserved (central and terminal stems) and nonconserved helices (hairpins 2, 3, and 4 [26]). The number of hairpins in this region varies from none to three in different organisms. The relative positions of the phylogenetically conserved sequence elements, boxes A, A', B, C, C', and D are well preserved in all U3 snoRNAs known. The box A' and A segments are believed to interact with pre-rRNA (see text). Box C influences association with the protein fibrillarin (6). Box D is required for formation of the 5' TMG cap structure and nuclear retention of mature RNA (68). The primary sequence of the hinge region separating the conserved elements of the 5' and 3' portions is not well preserved phylogenetically. Ten nucleotides of the hinge segment in yeast U3 have been postulated to interact with the 5' ETS of pre-rRNA through complementary base-pairing (8).

Functional mapping of U3 is still at an early stage. A few elements have been tied to different aspects of snoRNA synthesis, localization, or function. Box A of yeast U3 was shown to be essential for yeast cell viability, based on substitution and deletion mutations (24, 44). This is one of the two regions proposed to base-pair with pre-rRNA, based on cross-linking data obtained with yeast and animal U3 RNAs (66, 72). Box A' has also been implicated in base-pairing with pre-rRNA, and mutation of this element has been found to affect yeast cell viability (44). The hinge region of yeast U3 has been proposed to interact directly with pre-rRNA through a segment of 10 complementary nucleotides (9). Consistent with binding, a mutation in this segment rescued a lethal rRNA mutation by providing complementarity to the mutant rRNA region (8). No functional information is yet available for box B and the central stem. Mutations in box C influence U3 association with fibrillarin in human cell extracts, and alterations in box D and the 3' terminal stem block cap formation in Xenopus U3 transcripts injected into oocytes (6, 68). Mutations in the latter elements also impair retention of U3 in the nucleus (68). In yeast cells a large deletion covering most of the hinge region, the proximal part of the 3' terminal stem, and box C' has been shown to abolish U3 accumulation (44).

The present investigation was undertaken to develop a map of essential regions in this vital RNA. The major aims of our study were to evaluate the importance of the conserved and nonconserved elements for U3 production and function in S. cerevisiae. The elements featured in our analysis included (i) the 5' segment encompassing conserved boxes A' and A and the nonconserved hinge region; (ii) conserved boxes B, C, C', and D; (iii) the conserved central and terminal stems, and (iv) the nonconserved hairpins in the highly structured 3' domain.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Strains and media. S. cerevisiae JH84 (leu2-3,12 ura3-52 his3- ade2-1 can1100 u3a UAS_GAL:U3A::URA3 U3B::LEU2) was kindly provided by John Hughes and was used as the test strain (a similar strain, JH44, is described in reference 25). Yeast cells were grown at 30°C unless otherwise specified. Transformants were grown initially on selective solid 0.67% yeast nitrogen base (YNB) medium containing 2% galactose. This allowed maintenance of the introduced plasmid and expression of U3 from the galactose-dependent genomic cassette; hence, even cells containing nonfunctional U3 genes on the plasmid were able to grow.

80 lacZ-M15

DNA and RNA manipulations. Plasmid DNA was prepared from E. coli with a boiling miniprep procedure, and total yeast RNA was isolated with a hot-phenol-glass bead procedure (33, 59).

Preparation of modified U3 coding sequences. The original U3A gene was isolated from plasmid pRex4A (kindly provided by John Hughes) as a blunt-ended HindIII fragment and subcloned into the blunt-ended SalI site of the pBluescript IISK() vector with a previously removed BstXI site to generate plasmid pBU3. PCR strategies to alter the U3 gene were performed with this plasmid (Fig. 2). Plasmids used for yeast transformation were prepared by cloning EcoRI-XhoI fragments carrying the wild-type and mutated U3 RNA genes from the pBluescript IISK() vector into the yeast shuttle vector pRS313 (64). The final plasmids were as follows: pRU3, wild-type U3; pRU3*, tagged U3; pRU3*(C':GC), substitution of the first G in box C' with C; pRU3*(C':AT), substitution of the first A in box C' with T; pRU3*(C:GC), substitution of the first G in box C with C; pRU3*(C:AT), substitution of the first A in box C with T; pRU3*(D:AT), substitution of the last A in box D with T; pRU3*(C:subst), substitution of the entire box C sequence CGATGA with GCTACT; pRU3*(B:subst), substitution of the entire box B sequence GAGTGAG with CTCACTC; pRU3*(t.st:P), substitution of the terminal stem proximal sequence ACTTG with TGGGC; pRU3*(t.st:D), substitution of the terminal stem distal sequence CAAGT with GCCCA; pRU3*(t.st:PD), substitution of both proximal and distal sequences of the terminal stem; pRU3*(c.st:P), substitution of the central stem proximal sequence CCTTTGTAGGG with GGAAACATGGG; pRU3*(c.st:D), substitution of the central stem distal sequence GGGTACAAATCC with CCCATGTTTTCC; pRU3*(c.st:PD), substitution of both proximal and distal sequences of the terminal stem; pRU3*(trunc), deletion from the 5' end to the base of the terminal stem; and pRU3*(del), deletion of hairpins 2, 3, and 4. 

View larger version (18K): [in this window] [in a new window]   FIG. 2.   U3 mutagenesis strategies. Different PCR strategies were used for creating U3 mutations, depending on the position and length of the mutant sequence and the number of sequences to be substituted. In each case two PCR amplification steps were involved. To introduce point mutations or make a short sequence substitutions, the strategies presented in panels A or B were used. Multiple mutations were created with the strategy presented in panel C. Substitution or deletion of large regions were done as depicted in panel D. Arrows with numbers correspond to the oligonucleotide primers. Short vertical lines with letters correspond to restriction sites. Open and closed boxes designate mutant and wild-type sequences, respectively.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Genetic system for functional mapping of yeast U3 RNA. To assess the importance of U3 structural elements, we developed a genetic system that features a S. cerevisiae JH84 test strain containing a galactose-dependent U3 gene in the genome (UAS_GAL:U3A). All mutations were introduced into a constitutively expressed U3 gene on the plasmid, called U3* (Fig. 3A; see also Materials and Methods). The RNA expressed from the U3* gene contains a unique hybridization "tag," which makes it possible to distinguish the products of this allele from RNA produced from the genomic locus. When designing this gene, we took into consideration the fact that hairpin 4 of the S. cerevisiae U3 RNA is the least phylogenetically conserved portion of the molecule and in most organisms (including the yeast Schizosaccharomyces pombe) is absent altogether and thus probably plays an unimportant role. Modifications were made in this stem-loop domain that changed the primary sequence slightly but left the hypothetical secondary structure intact (Fig. 3B).

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Genetic system for functional mapping of yeast U3 RNA. (A) Mutational analyses were carried out with test strain JH84 (25). In this strain one of two genes encoding U3 RNA (U3B) is disrupted with a LEU2 marker gene, and the second (U3A) is under control of the UASGAL-regulated promoter. Cells grow well on medium containing galactose as the sole carbon source, since U3A RNA is transcribed from the promoter induced by galactose. In the presence of glucose the UASGAL-regulated promoter is repressed, and this leads to severe underaccumulation of U3 and subsequent cell death. Lethality can be avoided, however, if the cells also contain a plasmid with a functional U3 RNA gene under control of the normal U3 promoter. (B) In order to distinguish RNAs expressed from the chromosome and the plasmid, a modified U3 gene with a unique hybridization tag (U3*) was constructed. The U3* RNA has a different sequence in the nonconserved hairpin 4 region; however, the secondary structure of the hairpin is preserved. The importance of specific U3 elements for RNA function and production was evaluated by transforming test cells with the plasmid-encoded mutant genes and examining cell growth on glucose and the accumulation of mutant U3 RNA on galactose. (C) Depletion of wild-type U3 produced from the galactose-dependent chromosomal allele during cell growth on glucose was evaluated by Northern blotting analysis. The test strain was transformed with plasmids encoding wild-type (pRU3) or tagged (pRU3*) U3. Total RNA was prepared from transformants grown overnight in liquid selective medium containing 5% glucose. A blot containing these RNAs was first probed with a radiolabeled oligonucleotide (C163) that recognizes both wild-type (U3) and tagged U3 (U3*; left panel) RNAs. The blot was washed and then probed with an oligonucleotide (SD14) that recognizes exclusively wild-type U3 (right panel). Depletion of wild-type U3 is nearly complete in cells transformed with pRU3* and grown on glucose.

U3 contains a structural homolog of the U14 box C/D terminal stem motif. In box C/D snoRNAs, boxes C (UGAUGA) and D (GUCUGA) are usually located near the 5' and 3' ends of the mature molecule, respectively. Structure and phylogenetic studies of S. cerevisiae U14 RNA revealed that box C, box D, and an adjoining terminal stem form a box C/D stem structural motif (Fig. 4A; see also references 3 and 23). Identical structures occur in folding models of U14 molecules from other organisms (several examples are presented). In yeast and Xenopus sp., each component of the motif has been shown to be essential for U14 accumulation (23, 78). Certain of the box C and D nucleotides are particularly important (Fig. 4, circled).

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Structural homologs of the box C/D stem motif in U3 and U14 snoRNAs. (A) Boxes C and D of U14 snoRNA together with adjoining inverted repeats form a conserved structure known as the box C/D stem motif. Each of the components of this motif is essential for U14 accumulation in S. cerevisiae (23). Box C and box D nucleotides important for RNA production are circled. (B) Boxes C' and D of U3 can also be arranged in a secondary structure similar to that of the U14 box C/D stem motif. As in U14 this hypothetical structure is phylogenetically conserved in all U3 homologs. The nucleotides essential for U14 accumulation are also preserved in U3 (circled).

Boxes C' and D, but not box C, are essential for U3 accumulation. If the box C or C' element (or both) of U3 is a functional homolog of U14 box C, the corresponding nucleotides vital for U14 accumulation should also be necessary for U3 RNA production. This prediction was examined by introducing two point mutations in boxes C and C' of U3* which are known to cause severe underaccumulation of U14 (Fig. 5A; see also Fig. 4).

View larger version (22K): [in this window] [in a new window]   FIG. 5.   A box C/D stem motif containing box C' rather than box C is important for U3 accumulation. (A) Single base substitutions were made in U3 boxes C and C' at positions known to be essential for the box C stabilizing function in U14. A point mutation was created in box D, which in U14 is known to lead to underaccumulation of snoRNA (23). A substitution mutation which replaces the entire box C was also prepared. (B) Effects of mutations on U3 RNA accumulation were assessed by Northern blotting analysis of total RNA isolated from galactose-grown cells. Levels of mutant U3* RNA were estimated with a probe specific to a hybridization tag (upper portion of panel). RNA loading was assessed with hybridization probes for U14 and U1 RNAs. Mutation effects on cell viability were determined by growing transformants on glucose-containing plates (lower portion of panel). Growth rates are indicated as follows: + corresponds to wild-type growth, and  represents no growth.

Both stems flanking boxes C' and D are required for U3 production. We propose the box C' and D elements in U3 comprise a structure similar to the U14 box C/D stem motif. If true, the inverted repeats flanking boxes C' and D must form a helix in vivo that is important for snoRNA accumulation. To test this, we separately substituted both the 5' proximal [pRU3*(t.st:P)] and 3' distal [pRU3*(t.st:D)] inverted repeats with noncomplementary sequences. An additional construct, pRU3*(t.st:PD), contained proximal and distal stem segments with reconstituted base-pairing possibilities but with different sequences (Fig. 6A). Transformants containing these alleles were analyzed for growth on glucose, and RNA was prepared from cells grown on galactose. RNA expressed from plasmid pRU3*(t.st:P) accumulated at a very low level on galactose and did not support normal growth on glucose medium [Fig. 6B, lane pRU3*(t.st:P)]. Although U3 expressed from the pRU3*(t.st:D) construct supported normal growth, the mutant RNA accumulated at a level lower than did the U3* control [lane pRU3*(t.st:D)]. Cells dependent on the mutant allele in which substitution mutations restored base-pairing grew on glucose as well as cells expressing wild-type U3*; however, the level of the mutant RNA expressed on galactose actually exceeded that of wild-type U3* [lanes pRU3*(t.st:PD) and pRU3*].

View larger version (25K): [in this window] [in a new window]   FIG. 6.   The terminal and central stems are required for stable RNA production. (A) The importance of the terminal and central stems flanking boxes C' and D was evaluated with mutations that disrupt the complementary interactions. Mutations which change the primary but preserve the secondary structures of these stems were also prepared. (B) Substitutions in either proximal (P) or distal (D) parts of the terminal stem (t.st) or central stem (c.st) lead to underaccumulation of U3 on galactose medium and impair cell growth on glucose. Substitutions in both proximal and distal parts (PD) which preserve complementarity rescue these effects. Hybridization probes for the Northern blotting analyses and symbols indicating cell growth on glucose are the same as in Fig. 5.

Box B is required for U3 function but not accumulation. To assess the importance of the conserved box B, we prepared a construct in which the seven most phylogenetically stable nucleotides were substituted with an unrelated sequence [pRU3*(B:subst); Fig. 7A]. Expression of this construct in test cells cultured on galactose revealed no change in accumulation level, indicating that box B is not important for U3 production (Fig. 7B). While the RNA accumulated, it could not support normal growth of cells on solid glucose medium. To assess the effect of the mutation in more detail, we analyzed cell growth at different temperatures and in liquid medium. At 15°C and on solid glucose medium, cells expressing U3*(B:subst) grew more slowly than those expressing wild-type U3*. However, at 37°C both types of cells grew equally well. A growth curve analysis at 30°C in liquid glucose medium revealed that the onset of logarithmic growth was delayed 10 h (data not shown). The basis of this effect is unknown. In any case, the cold-sensitive growth indicates that box B is important for U3 RNA function but does not affect its accumulation.

View larger version (22K): [in this window] [in a new window]   FIG. 7.   Box B is required for U3 function but not RNA accumulation. (A) The importance of the highly conserved box B element was examined by substituting the entire box with an unrelated sequence. (B) Substitution of box B (B:subst) does not affect U3 accumulation on galactose but does cause a cs slow-growth phenotype for cells grown on glucose medium. Probes and growth symbols are the same as in Fig. 5, except that ± refers to partially impaired growth (see text).

U3 lacking a 5' segment is toxic, but mutation of box C rescues this effect. The box A' and A elements of the U3 5' segment are known to be required for RNA function but not RNA stability (24, 44). We evaluated a construct in which nearly all of the 5'-end segment was removed, leaving only six native nucleotides at the very 5' end of the molecule and one nucleotide preceding the proximal part of the terminal stem [pRU3*(trunc); Fig. 8A]. The truncated RNA accumulated normally on galactose but was unable to support growth on glucose medium [Fig. 8B, lane pRU3*(trunc)]. Interestingly, cells containing the abnormal RNA grew more slowly on galactose-containing plates than cells containing U3* or just the plasmid vector pRS313. The growth curve of cells cultured in liquid galactose medium revealed a pre-logarithmic-phase delay of 8 h (not shown). We wondered if the toxic effect of the truncated RNA might reflect competition of this molecule for an essential trans-acting factor(s). Results from this and earlier studies suggest that a factor(s), presumably a protein(s), binds to the central part of U3 where the conserved boxes B and C occur (5, 6, 37, 44, 50, 68). The possibility that the truncated U3 variant competes for such a factor(s) was examined with a mutant U3 containing the same deletion, as well as with a fully substituted box C mutation (Fig. 8A). Strikingly, the box C mutation rescued both the slow-growth phenotype on solid galactose medium and the delayed-growth phenotype in liquid medium (not shown). The mutant RNA was produced at a normal level [Fig. 8B, lane pRU3*(trunc/C:subst)].

View larger version (24K): [in this window] [in a new window]   FIG. 8.   U3 lacking the 5' segment is toxic, but substitution of box C rescues this effect. (A) The importance of the entire 5' segment for U3 RNA accumulation was examined by creating a deletion spanning the region between position +6 and a nucleotide preceding the terminal stem. (B) This variant, U3*(trunc), accumulates normally in cells grown in galactose but, as expected, does not support growth on glucose. Interestingly, cells expressing both the wild-type and truncated forms of U3 on galactose grow slower than cells which only express normal U3. Substitution of box C with an unrelated sequence, U3*(trunc/C:subst), rescues the toxic effect of the truncated molecule in cells grown on galactose. This fact suggests binding of the hypothetical trans-acting factor(s) with the portion of U3 where boxes B and C are located. Probes and growth symbols are as in Fig. 5 and 7.

The essential 5' region contains a previously unidentified GAC box element. To gain additional insight into the role of the 5' region, we conducted a phylogenetic comparison of the 5' segments from more than 20 U3 RNA species (Fig. 9). This analysis revealed a novel, absolutely conserved GAC box element which precedes box A'. The conserved nature of this box suggests that it is important for U3 function (see also Discussion).

View larger version (69K): [in this window] [in a new window]   FIG. 9.   Phylogenetic comparison of U3 5' regions. Alignment of the 5' segment sequences from diverse organisms reveals a novel conserved GAC box element. The length but not the primary structure of the hinge region appears well preserved. The U3 sequences analyzed (GenBank accession numbers are given in parentheses) are from protists Leptomonas collosoma (L32919), Trypanosoma brucei (M25776), Leishmania tarentolae (L20948), Tetrahymena thermophila (X71349), Euglena gracilis (U27297), and Dictyostelium discoideum (V00190); fungi Saccharomyces cerevisiae (M26648 and X05498), Hansenula wingei (X91005), and Schizosaccharomyces pombe (X56982 and X56189); plants Zea mays (Z29641), Oryza sativa (X79685), Triticum aestivum (X63065), Solanum lycopersicum (X14411), Solanum tuberosum (Z11883), and Arabidopsis thaliana (X52629, X52630, and X58068); and animals Xenopus laevis (X07318), Xenopus borealis (X07319), Mus musculus (X63743), Rattus norvegicus (J01884 and K00780), and Homo sapiens (M14061). The first nucleotide (R) follows the cap (either TMG or gamma-monomethyl phosphate). T.st(P) corresponds to the proximal region of the terminal stem. The G* nucleotide in the GAC box of mouse U3 has been shown to cross-link to the 5' ETS of pre-rRNA (72).

Hairpins 2, 3, and 4 are not required for accumulation or function. The functional importance of the nonconserved hairpins 2, 3, and 4 was evaluated with a deletion construct that removed all three stem-loop structures [pRU3*(del); Fig. 10A]. In this construct the hairpins were almost completely deleted, leaving only the nucleotides which comprise the first base pair of each hairpin. Surprisingly, the RNA expressed from the new allele was stable and also provided normal growth in the absence of wild-type U3 (Fig. 10B). Minor larger species present in the Northern blot correspond to U3 derivatives with a 3' end extended by approximately 20 to 30 nucleotides, as demonstrated with hybridization probes specific for the 5' or 3' flanking regions in the U3 gene (not shown). These striking results indicate that in yeast cells a small U3 variant lacking most of the nonconserved sequence is able to accumulate normally and provide all of the essential functions. This mini-variant resembles the U3 RNAs from protists (18, 21, 49).

View larger version (20K): [in this window] [in a new window]   FIG. 10.   Hairpins 2, 3, and 4 together are not essential for U3 accumulation and function. (A) The importance of hairpins 2, 3, and 4 was evaluated by deleting all three stem-loop domains. (B) The ability of mutant U3(del) RNA to accumulate was assessed with cells grown in liquid galactose medium, in which natural U3 is also produced from the chromosomal gene. Since the mutant RNA lacks hairpin 4, which contains the unique hybridization tag, a different probe was used in this analysis. This probe (SD74) hybridizes to the U3(del) region corresponding to the central stem and boxes B and C, which are also present in the wild-type U3 produced from the genomic locus. Since U3(del) is much smaller than wild-type U3 it is easily identified. The mutant U3(del) accumulates at a good level on galactose and, remarkably, supports wild-type growth of cells on glucose medium, where the wild-type U3 gene is repressed. Probes for control RNAs and growth symbols are the same as in Fig. 5.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Our results provide several new insights into the structure and function elements of the essential U3 snoRNA from S. cerevisiae and should be relevant to other U3 homologs as well. The genetic system used allowed us not only to identify defective U3 genes but also to distinguish mutations that influence U3 production from others that affect only U3 function.

The unfolded 5' segment of U3 is essential for activity but not RNA production. Deleting most of the 5' portion of U3 did not affect RNA accumulation; rather, a stable, but nonfunctional by-product was produced. Biochemical and genetic studies conducted previously with U3 RNAs from various organisms strongly suggest that the 5' segment interacts directly with pre-rRNA (see the introduction). However, attempts to identify specific nucleotides involved in such interaction(s) have yielded ambiguous results. The primary candidates in U3 are the highly conserved nucleotides in the box A'/A region, and the proposed target sites in pre-rRNA include nucleotides in the noncoding region of the 5' ETS and in an early portion of the 18S rRNA coding region (9, 24, 44, 66, 72). In reality U3 may interact with pre-rRNA at multiple sites, perhaps in an evolving dynamic fashion.

Boxes C' and D comprise a recognition motif characteristic of other box C/D snoRNAs. Previous studies with other box C/D snoRNAs revealed that box C, box D, and an adjoining terminal stem are required for snoRNA accumulation and form a structural motif in mature RNA (3, 12, 13, 23, 76, 78). This box C/D stem motif is believed to be recognized by a hypothetical protein(s) which protects the mature RNA from exonucleolytic degradation. Our mutational results demonstrate that boxes C' and D and flanking complementary elements are required for U3 production, as shown earlier for boxes C and D and flanking inverted repeats of yeast U14 (see also reference 23). These results argue that the box C'/D stem motif of U3 corresponds functionally to the box C/D motif of U14. Consistent with this view and a role in protein binding, the box C'/D stem segments were found to be protected in structure probing studies of U3 RNP complexes in human and yeast cell extracts and in intact yeast cells (44, 50). The canonical box C is located far from box D in the U3 secondary structure. This fact and our demonstration that box C is not required for RNA production indicate that box C in U3 is not homologous to box C from other box C/D snoRNAs.

Boxes B and C are predicted to form a recognition motif for a U3-specific protein(s). The canonical box C element is required for normal function of U3; this is due, most likely, to interaction with some trans-acting factor(s). In the secondary structure, box B is always found in close proximity to box C, suggesting that boxes B and C may form a recognition motif and interact with the same factor(s). The importance of box B was demonstrated by a substitution mutation which impaired U3 function, although to a lesser degree than substitution of box C.

Nearly 50% of wild-type U3 is dispensable. Our results demonstrate that the nonconserved stem-loop domains in the highly structured 3' portion are not required for U3 accumulation or function. Molecules lacking all three hairpins, corresponding to about 50% of the nucleotides, accumulate and provide normal function. This fact, though quite remarkable, is not completely surprising. For example, the yeast U2 spliceosomal RNA is almost six times larger than mammalian U2 and more than 80% of the molecule, corresponding to the nonconserved parts, can be removed without significantly affecting function (1, 63). The U3 from S. cerevisiae with 333 nucleotides is among the largest known U3 molecules. The smallest ones, from protists, are about 50% smaller and do not possess hairpins 2 to 4 (18, 21, 49). However, the latter RNAs contain all of the phylogenetically conserved U3 elements. Our functional mini-U3 molecule looks very similar to the protist homologs.

Structure-function map of U3. The structural and functional information available for the U3 small nucleolar RNA from S. cerevisiae is summarized in Fig. 11. The 5' segment containing the conserved GAC, A', and A boxes is believed to be involved in direct interaction with pre-rRNA, which is critical for rRNA processing. The hinge region is proposed to serve a spacer function to divide the protein-covered 3' portion of U3 and the 5' segment, which interacts with pre-rRNA. The 5' TMG cap is postulated to protect the 5' region upstream of the box C'/D stem motif from exonucleolytic degradation. The box C'/D stem and box B/C motifs are proposed to be recognition signals for RNA binding proteins. A protein(s) associated with the box C'/D stem motif provides U3 stability and processing and allows nucleolar targeting functions. Protein(s) recognizing the box B/C motif is predicted to be important for U3 function. Finally, the nonconserved hairpins 2, 3, and 4 are dispensable but appear to facilitate RNA folding and processing, thus affecting the homogeneity of the final U3 products. The present map will be useful for studies aimed at identifying the precise role that U3 RNA plays in ribosome biogenesis and characterizing its mechanism of action.

View larger version (44K): [in this window] [in a new window]   FIG. 11.   Functional map of U3 snoRNA. Results from the present and earlier studies have been combined to yield a functional map of the U3 molecule from S. cerevisiae. The 5' segment encompassing conserved boxes A and A' and a novel conserved element, designated the GAC box, is involved in direct interaction with pre-rRNA and is essential for snoRNA function but not production. A hinge region is predicted to provide proper spacing between the 5' portion which interacts with pre-rRNA and the 3' segment complexed with RNA binding proteins. The 5' and hinge segments are protected from degradation by a TMG (gamma-monomethyl phosphate in plants) cap. The box C'/D stem motif is homologous to the box C/D stem motifs in other box C/D snoRNAs and most likely serves as a recognition element for protein(s) which in U3 (i) protects the RNA from degradation, (ii) facilitates formation of the 5' TMG-cap, and (iii) specifies nucleolar localization. Formation of the box C'/D stem structure requires pairing of the central stem. Boxes B and C are proposed to form a box B/C structure motif which serves as a recognition element for a trans-acting factor(s), presumably a protein(s). Interaction with this factor(s) is required for U3 function. Hairpins 2, 3, and 4 are dispensable for U3 accumulation and function. These latter domains may be considered a species-specific "signature." The nucleotide regions corresponding to the minimal functional U3 are indicated by boldface letters. The shaded areas correspond to putative protein recognition sites.