Metal resistance in yeast mediated by the expression of a maize 20S proteasome alpha subunit

A novel 72 nt small nucleolar RNA (snoRNA) called U87 was found in rat liver cells. This RNA possesses the features of C/D box snoRNA family: boxes C, D', C', D, and 11 nt antisense element complementary to 28S ribosomal RNA (rRNA). The vast majority of C/D box snoRNAs direct site-specific 2'-O-ribose methylation of rRNAs. U87 RNA is suggested to be involved in 2'-O-methylation of a G(3468) residue in 28S rRNA. U87 RNA was detected in different mammalian species with slight length variability. Rat and mouse U87 RNA gene was characterized. Unlike the majority of C/D box snoRNAs U87 RNA lacks the terminal stem required for snoRNA processing. However, U87 gene is flanked by 7 bp inverted repeats potentially able to form a terminal stem in U87 RNA precursor.     

Identification of a new ribose methylation in the 18S rRNA of S. cerevisiae

Methylation of ribose sugars at the 2′-OH group is one of the major chemical modifications in rRNA, and is catalyzed by snoRNA directed C/D box snoRNPs. Previous biochemical and computational analyses of the C/D box snoRNAs have identified and mapped a large number of 2′-OH ribose methylations in rRNAs. In the present study, we systematically analyzed ribose methylations of 18S rRNA in Saccharomyces cerevisiae, using mung bean nuclease protection assay and RP-HPLC. Unexpectedly, we identified a hitherto unknown ribose methylation at position G562 in the helix 18 of 5′ central domain of yeast 18S rRNA. Furthermore, we identified snR40 as being responsible to guide snoRNP complex to catalyze G562 ribose methylation, which makes it only second snoRNA known so far to target three ribose methylation sites: Gm562, Gm1271 in 18S rRNA, and Um898 in 25S rRNA. Our sequence and mutational analysis of snR40 revealed that snR40 uses the same D′ box and methylation guide sequence for both Gm562 and Gm1271 methylation. With the identification of Gm562 and its corresponding snoRNA, complete set of ribose methylations of 18S rRNA and their corresponding snoRNAs have finally been established opening great prospects to understand the physiological function of these modifications.     

U86, a novel snoRNA with an unprecedented gene organization in yeast

The Xenopus laevis Nop56 gene (XNOP56), coding for a snoRNP-specific factor, belongs to the 5'-TOP gene family. XNOP56, as many 5'-TOP genes, contains an intron-encoded snoRNA. This previously unidentified RNA, named U86, was found as a highly conserved species in yeast and human. While in human it is also encoded in an intron of the hNop56 gene, in yeast it has an unprecedented gene organization: it is encoded inside an open-reading frame. Both in X. laevis and yeast, the synthesis of U86 snoRNA appears to be alternative to that of the cotranscribed mRNA. Despite the overall homology, the three U86 snoRNAs do not show strong conservation of the sequence upstream from the box D and none of them displays significant sequence complementarity to rRNA or snRNA sequences, suggesting a role different from that of methylation.     

Box C/D Small Nucleolar RNA (snoRNA) U60 Regulates Intracellular Cholesterol Trafficking

Mobilization of plasma membrane (PM) cholesterol to the endoplasmic reticulum is essential for cellular cholesterol homeostasis. The mechanisms regulating this retrograde, intermembrane cholesterol transfer are not well understood. Because mutant cells with defects in PM to endoplasmic reticulum cholesterol trafficking can be isolated on the basis of resistance to amphotericin B, we conducted an amphotericin B loss-of-function screen in Chinese hamster ovary (CHO) cells using insertional mutagenesis to identify genes that regulate this trafficking mechanism. Mutant line A1 displayed reduced cholesteryl ester formation from PM-derived cholesterol and increased de novo cholesterol synthesis, indicating a deficiency in retrograde cholesterol transport. Genotypic analysis revealed that the A1 cell line contained one disrupted allele of the U60 small nucleolar RNA (snoRNA) host gene, resulting in haploinsufficiency of the box C/D snoRNA U60. Complementation and mutational studies revealed the U60 snoRNA to be the essential feature from this locus that affects cholesterol trafficking. Lack of alteration in predicted U60-mediated site-directed methylation of 28 S rRNA in the A1 mutant suggests that the U60 snoRNA modulates cholesterol trafficking by a mechanism that is independent of this canonical function. Our study adds to a growing body of evidence for participation of small noncoding RNAs in cholesterol homeostasis and is the first to implicate a snoRNA in this cellular function.     

Fibrillarin genes encode both a conserved nucleolar protein and a novel small nucleolar RNA involved in ribosomal RNA methylation in Arabidopsis thaliana

Fibrillarin is a key nucleolar protein in eukaryotes which associates with box C/D small nucleolar RNAs (snoRNAs) directing 2'-O-ribose methylation of the rRNA. In this study we describe two genes in Arabidopsis thaliana, AtFib1 and AtFib2, encoding nearly identical proteins conserved with eukaryotic fibrillarins. We demonstrate that AtFib1 and AtFib2 proteins are functional homologs of the yeast Nop1p (fibrillarin) and can rescue a yeast NOP1-null mutant strain. Surprisingly, for the first time in plants, we identified two isoforms of a novel box C/D snoRNA, U60.1f and U60.2f, nested in the fifth intron of AtFib1 and AtFib2. Interestingly after gene duplication the host intronic sequences completely diverged, but the snoRNA was conserved, even in other crucifer fibrillarin genes. We show that the U60f snoRNAs accumulate in seedlings and that their targeted residue on the 25 S rRNA is methylated. Our data reveal that the three modes of expression of snoRNAs, single, polycistronic, and intronic, exist in plants and suggest that the mechanisms directing rRNA methylation, dependent on fibrillarin and box C/D snoRNAs, are evolutionarily conserved in plants.     

Functional importance of individual rRNA 2'-O-ribose methylations revealed by high-resolution phenotyping

Ribosomal RNAs contain numerous modifications at specific nucleotides. Despite their evolutionary conservation, the functional role of individual 2'-O-ribose methylations in rRNA is not known. A distinct family of small nucleolar RNAs, box C/D snoRNAs, guides the methylating complex to specific rRNA sites. Using a high-resolution phenotyping approach, we characterized 20 box C/D snoRNA gene deletions for altered growth dynamics under a wide array of environmental perturbations, encompassing intraribosomal antibiotics, inhibitors of specific cellular features, as well as general stressors. Ribosome-specific antibiotics generated phenotypes indicating different and long-ranging structural effects of rRNA methylations on the ribosome. For all studied box C/D snoRNA mutants we uncovered phenotypes to extraribosomal growth inhibitors, most frequently reflected in alteration in growth lag (adaptation time). A number of strains were highly pleiotropic and displayed a great number of sensitive phenotypes, e.g., deletion mutants of snR70 and snR71, which both have clear human homologues, and deletion mutants of snR65 and snR68. Our data indicate that individual rRNA ribose methylations can play either distinct or general roles in the workings of the ribosome.     

A novel snoRNA gene cluster in yeast is transcribed as polycistronic pre-snoRNAs

Small nucleolar RNAs (snoRNAs) play an important role in eukaryotic rRNA biogenesis. By combination of a computer search of EMBL database and experimental procedure, a novel snoRNA coding sequence (Z8) was screened out and characterized from yeastSaccharomyces cerevisiue genome. Z8 snoRNA gene codes a boxC/D antisense snoRNA which guides, deduced from structure analysis, the 2′-O-ribose methylation at U₂₄₂₁ of 25S rRNA. After disruption of Z8 snoRNA gene, the methylation at corresponding site was abolished, but no gmwth delay was observed in various cultural temperatures. Z8 DNA is the first gene of a gene cluster consisting of three cognate snoRNA genes which are located on an intergenic region of chromosome XIII. This gene cluster is co-transcribed as a polycistronic precursor from a + 247 bp U snoRNA gene promoter, followed by processing to release individual snoRNAs, representing a new expression pattern of snoRNA genes.     

A novel snoRNA gene cluster in yeast is transcribed as polycistronic pre-snoRNAs

Small nueleolar RNAs (snoRNAs) play an important role in eukaryotic rRNA biogenesis. By combination of a computer search of EMBL database and experimental procedure, a novel snoRNA coding sequence (Z8) was screened out and characterized from yeast Saccharomyces cerevisiae genome. Z8 snoRNA gene codes a boxC/D antisonse snoRNA which guides, deduced from structure analysis, the 2'-O-ribose methylation at U_(2421) of 25S rRNA. After disruption of Z8 snoRNA gene, the methylation at corresponding site was abolished, but no growth delay was observed in various cultural temperatures. Z8 DNA is the first gene of a gene cluster consisting of three cognate snoRNA genes which are located on an intergenie region of chromosome ⅩⅢ. This gene cluster is co-transcribed as a pelycistronic precursor from a 247 bp U snoRNA gene promoter, followed by processing to release individual snoRNAs, representing a new expression pattern of snoRNA genes.     

The high diversity of snoRNAs in plants: identification and comparative study of 120 snoRNA genes from Oryza sativa

Using a powerful computer-assisted analysis strategy, a large-scale search of small nucleolar RNA (snoRNA) genes in the recently released draft sequence of the rice genome was carried out. This analysis identified 120 different box C/D snoRNA genes with a total of 346 gene variants, which were predicted to guide 135 2′-O-ribose methylation sites in rice rRNAs. Though not exhaustive, this analysis has revealed that rice has the highest number of known box C/D snoRNAs among eukaryotes. Interestingly, although many snoRNA genes are conserved between rice and Arabidopsis, almost half of the identified snoRNA genes are rice specific, which may highlight further the differences in rRNA methylation patterns between monocotyledons and dicotyledons. In addition to 76 singletons, 70 clusters involving 270 snoRNA genes were also found in rice. The large number of the novel snoRNA polycistrons found in the introns of rice protein-coding genes is in contrast to the one-snoRNA-per-intron organization of vertebrates and yeast, and of Arabidopsis in which only a few intronic snoRNA gene clusters were identified. Furthermore, due to a high degree of gene duplication, rice snoRNA genes are clearly redundant and exhibit great sequence variation among isoforms, allowing generation of new snoRNAs for selection. Thus, the large snoRNA gene family in plants can serve as an excellent model for a rapid and functional evolution.     

Identification of a novel box C/D snoRNA from mouse nucleolar cDNA library

By construction and screen of mouse nucleolar cDNA library, a novel mammalian small nucleolar RNAs (snoRNA) was identified. The novel snoRNA, 70 nt in length, displays structural features typical of C/D box snoRNA family. The snoRNA possesses an 11-nt-long rRNA antisense element and is predicted to guide the 2'-O-methylation of mouse 28S rRNA at G4043, a site unknown so far to be modified in vertebrates. The comparison of functional element of snoRNA guides among eukaryotes reveals that the novel snoRNA is a mammalian counterpart of yeast snR38 despite highly divergent sequence between them. Mouse and human snR38 and other cognates in distant vertebrates were positively detected with slight length variability. As expected, the rRNA ribose-methylation site predicted by mouse snR38 was precisely mapped by specific-primer extension assay. Furthermore, our analyses show that mouse and human snR38 gene have multiple variants and are nested in the introns of different host genes with unknown function. Thus, snR38 is a phylogenetically conserved methylation guide but exhibits different genomic organization in eukaryotes.     

Sequence and structural elements of methylation guide snoRNAs essential for site-specific ribose methylation of pre-rRNA.

Site-specific 2'-O-ribose methylation of eukaryotic rRNAs is guided by small nucleolar RNAs (snoRNAs). The methylation guide snoRNAs carry long perfect complementaries to rRNAs. These antisense elements are located either in the 5' half or in the 3' end region of the snoRNA, and are followed by the conserved D' or D box motifs, respectively. An uninterrupted helix formed between the rRNA and the antisense element of the snoRNA, in conjunction with the adjacent D' or D box, constitute the recognition signal for the putative methyltransferase. Here, we have identified an additional essential box element common to methylation guide snoRNAs, termed the C' box. We show that the C' box functions in concert with the D' box and plays a crucial role in the methyltransfer reaction directed by the upstream antisense element and the D' box. We also show that an internal fragment of U24 methylation guide snoRNA, encompassing the upstream antisense element and the D' and C' box motifs, can support the site-specific methylation of rRNA. This strongly suggests that the C box of methylation guide snoRNAs plays an essential role in the methyltransfer reaction guided by the 3'-terminal antisense element and the D box of the snoRNA.     

Exploration of pairing constraints identifies a 9 base-pair core within box C/D snoRNA-rRNA duplexes

2'-O-ribose methylation of eukaryotic ribosomal RNAs is guided by RNA duplexes consisting of rRNA and box C/D small nucleolar (sno)RNA sequences, the methylated sites invariably mapping five positions apart from the D box. Here we have analyzed the RNA duplex pairing constraints by investigating the features of 415 duplexes from the fungus, plant and animal kingdoms, and the evolution of those duplexes within the 124 sets they group into. The D-box upstream 1st and >or=15th positions consist of Watson-Crick base-pairs, G:U base-pairs and mismatched bases with ratios close to random assortments; these positions display single base differences in >60% of the RNA duplex sets. The D-box upstream 2nd to 11th positions have >90% Watson-Crick base-pairs; they display single base mutations with a U-shaped distribution of lower values of 0% and 1.6% at the methylated site 5th and 4th positions, and double compensatory mutations leading to new Watson-Crick base-pairs with an inverted U-shaped distribution of higher values at the 8th to 11th positions. Half of the single mutations at the 3rd to 11th positions resulted in G:U base-pairing, mainly through A-->G mutations in the rRNA strands and C-->T mutations in the snoRNA strands. Double compensatory mutations at the 3rd to 11th positions are extremely frequent, representing 36% of all mutations; they frequently arose from an A-->G mutation in the rRNA strands followed by a T-->C mutation in the snoRNA strands. Differences in the mutational pathways through which the rRNA and snoRNA strand evolved must be related to differences in the rRNA and snoRNA copy number and gene organization. Altogether these data identify the D-box upstream 3rd to 11th positions as box C/D snoRNA-rRNA duplex cores. The impact of the pairing constraints on the evolution of the 9 base-pair RNA duplex cores is discussed.     

A novel snoRNA can direct site-specific 2'-O-ribose methylation of snRNAs in Oryza sativa

Small nucleolar RNAs (snoRNAs) are a kind of noncoding RNAs, and the vast majority of snoRNAs are involved in site-specific modifications of rRNAs. A novel box C/D snoRNA called snoR124 was found inOryza sativa, and it can direct 2'-O-ribose methylation of spliceosomal small nuclear RNAs (snRNAs). The snoRNA has two antisense elements, and the results of primer extensions at different dNTP concentrations provide evidence that snoR124 guide 2'-O-methylations of the C76 residue in the U4 snRNA and the T91 residue in the U5 snRNA. In addition, this snoRNA is located in a snoRNA gene cluster with another 7 snoRNAs which are identified to direct ribose methylations in rRNAs. This is consistent with the opinion that the snoRNA gene organization in plant is mainly gene cluster. The snoR124 is the first example of a snoRNA that directs modifications of RNAs other than rRNAs in plant; it will avail to get more insights into the function of snoRNAs in plant.     

SnoRNA-guided ribose methylation of rRNA: structural features of the guide RNA duplex influencing the extent of the reaction.

Eukaryotic rRNAs contain a large number of ribose-methylated nucleotides of elusive function which are confined to the universally conserved rRNA domains. Ribose methylation of these nucleotides is directed by a large family of small trans -acting guide RNAs, called box C/D antisense snoRNAs. Each snoRNA targets precisely one of the nucleotides to be methylated within the pre-rRNA sequence, through transient formation of a 10-21 bp regular RNA duplex around the modification site. In this study we have analyzed how different features of the double-stranded RNA guide structure affect the extent of site-specific ribose methylation, by co-expressing an appropriate RNA substrate and its cognate tailored snoRNA guide in transfected mouse cells. We show that an increased GC content of the duplex can make up for the inhibitory effects of a helix truncation or for the presence of helix irregularities such as a mismatched pair or a bulge nucleotide. However, some helix irregularities dramatically inhibit the reaction and are not offset by further stabilization of the duplex. Overall, the RNA duplex tolerates a much larger degree of irregularity than anticipated, even in the immediate vicinity of the methylation site, which offers new prospects in the search for additional snoRNA guides. Accordingly, a few snoRNA-like sequences of uncertain status detected in the yeast Saccharomyces cerevisiae genome now appear as likely bona fide ribose methylation guides.