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.     

Interference probing of rRNA with snoRNPs: a novel approach for functional mapping of RNA in vivo

Synthesis of eukaryotic ribosomal RNAs (rRNAs) includes methylation of scores of nucleotides at the 2'-O-ribose position (Nm) by small nucleolar RNP complexes (snoRNPs). Sequence specificity is provided by the snoRNA component through base-pairing of a guide sequence with rRNA. Here, we report that methylation snoRNPs can be targeted to many new sites in yeast rRNA, by providing the snoRNA with a novel guide sequence, and that in some cases growth and translation activity are strongly impaired. Novel snoRNAs can be expressed individually or by a unique library strategy that yields guide sequences specific for a large target region. Interference effects were observed for sites in both the small and large subunits, including the reaction center region. Targeting guide RNAs to nucleotides flanking the sensitive sites caused little or no defect, indicating that methylation is responsible for the interference rather than a simple antisense effect or misguided chaperone function. To our knowledge, this is the only approach that has been used to mutagenize the backbone of rRNA in vivo.     

Fibrillarin-associated box C/D small nucleolar RNAs in Trypanosoma brucei. Sequence conservation and implications for 2'-O-ribose methylation of rRNA

We report the identification of 17 box C/D fibrillarin-associated small nucleolar RNAs (snoRNAs) from the ancient eukaryote, Trypanosoma brucei. To systematically isolate and characterize these snoRNAs, the T. brucei cDNA for the box C/D snoRNA common protein, fibrillarin, was cloned and polyclonal antibodies to the recombinant fibrillarin protein were generated in rabbits. Immunoprecipitations from T. brucei extracts with the anti-fibrillarin antibodies indicated that this trypanosomatid has at least 30 fibrillarin-associated snoRNAs. We have sequenced seventeen of them and designated them TBR for T. brucei RNA 1-17. All of them bear conserved box C, D, C', and D' elements, a hallmark of fibrillarin-associated snoRNAs in eukaryotes. Fourteen of them are novel T. brucei snoRNAs. Fifteen bear potential guide regions to mature rRNAs suggesting that they are involved in 2'-O-ribose methylation. Indeed, eight ribose methylations have been mapped in the rRNA at sites predicted by the snoRNA sequences. Comparative genomics indicates that six of the seventeen are the first trypanosome homologs of known yeast and vertebrate methylation guide snoRNAs. Our results indicate that T. brucei has many fibrillarin-associated box C/D snoRNAs with roles in 2'-O-ribose methylation of rRNA and that the mechanism for targeting the nucleotide to be methylated at the fifth nucleotide upstream of box D or D' originated in early eukaryotes.     

Structural features of the guide:target RNA duplex required for archaeal box C/D sRNA-guided nucleotide 2'-O-methylation

Archaeal box C/D sRNAs guide the 2'-O-methylation of target nucleotides using both terminal box C/D and internal C'/D' RNP complexes. In vitro assembly of a catalytically active Methanocaldococcus jannaschii sR8 box C/D RNP provides a model complex to determine those structural features of the guide:target RNA duplex important for sRNA-guided nucleotide methylation. Watson-Crick pairing of guide and target nucleotides was found to be essential for methylation, and mismatched bases within the guide:target RNA duplex also disrupted nucleotide modification. However, dependence upon Watson-Crick base-paired guide:target nucleotides for methylation was compromised in elevated Mg(2+) concentrations where mismatched target nucleotides were modified. Nucleotide methylation required that the guide:target duplex consist of an RNA:RNA duplex as a target ribonucleotide within a guide RNA:target DNA duplex that was not methylated. Interestingly, D and D' target RNAs exhibited different levels of methylation when deoxynucleotides were inserted into the target RNA or when target methylation was carried out in elevated Mg(2+) concentrations. These observations suggested that unique structural features of the box C/D and C'/D' RNPs differentially affect their respective methylation capabilities. The ability of the sR8 box C/D sRNP to methylate target nucleotides positioned within highly structured RNA hairpins suggested that the sRNP can facilitate unwinding of double-stranded target RNAs. Finally, increasing target RNA length to extend beyond those nucleotides that base pair with the sRNA guide sequence significantly increased sRNP turnover and thus nucleotide methylation. This suggests that target RNA interaction with the sRNP core proteins is also important for box C/D sRNP-guided nucleotide methylation.     

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.     

Site-specific cross-linking analyses reveal an asymmetric protein distribution for a box C/D snoRNP

Methylation of the ribose 2'-hydroxyl, the most widespread modification of ribosomal and splicesomal RNAs, is guided by the box C/D class of small nucleolar RNAs (snoRNAs). Box C/D small nucleolar ribonucleoproteins (snoRNPs) contain four core proteins: fibrillarin, Nop56, Nop58 and 15.5 kDa. We constructed U25 snoRNAs containing a single photoactivatable 4-thiouridine at each U position within the conserved box C/D and C'/D' motifs. Proteins assembled on the snoRNA after injection into Xenopus oocyte nuclei were identified by cross-linking, and reconstituted particles characterized by functional rescue and mutational analyses. Our data argue that box C/D snoRNPs are asymmetric, with the C' box contacting Nop56 and fibrillarin, the C box interacting with Nop58, and the D and D' boxes contacting fibrillarin. No cross-link to 15.5 kDa was detected; its binding is disrupted by 4-thiouridine substitution in position 1 of the C box. Repositioning the guide sequence of U25 upstream of box D instead of D' revealed that both C/D motifs have the potential to function as guide centers, but, surprisingly, there was no alteration in protein cross-linking.     

snoRNA的结构与功能

核仁小分子RNA（snoRNA）是一类广泛分布于真核生物细胞核仁的小分子非编码RNA,具有保守的结构元件,并以此划分为3大类：boxC／DsnoRNA、boxH／ACAsnoRNA和MRPRNA。其中boxC／D和boxH／ACA是已知snoRNA的主要类型,以碱基配对的方式分别指导着核糖体RNA的甲基化和假尿嘧啶化修饰。研究发现,snoRNA除了在核糖体RNA的生物合成中发挥作用之外,还能够指导snRNA、tRNA和mRNA的转录后修饰。此外,还有相当数量的snoRNA功能不明,被称为孤儿sn0RNA（orphansnoRNA）。在哺乳动物的孤儿snoRNA中,印迹snoRNA（imprintedsnoRNA）是最为特殊的一群,由基因组印迹区编码,具有明显的组织表达特异性。原核生物古细菌中类snoRNA的鉴定表明这些非编码RNA家族成员的古老起源;而哺乳动物中大量的snoRNA反转座子的存在更为人们探索snoRNA在基因组中扩增和功能进化提供了新的思路。     

Conserved boxes C and D are essential nucleolar localization elements of U14 and U8 snoRNAs.

Sequences necessary for nucleolar targeting were identified in Box C/D small nucleolar RNAs (snoRNAs) by fluorescence microscopy. Nucleolar preparations were examined after injecting fluorescein-labelled wild-type and mutated U14 or U8 snoRNA into Xenopus oocyte nuclei. Regions in U14 snoRNA that are complementary to 18S rRNA and necessary for rRNA processing and methylation are not required for nucleolar localization. Truncated U14 molecules containing Boxes C and D with or without the terminal stem localized efficiently. Nucleolar localization was abolished upon mutating just one or two nucleotides within Boxes C and D. Moreover, the spatial position of Boxes C or D in the molecule is essential. Mutations in Box C/D of U8 snoRNA also impaired nucleolar localization, suggesting the general importance of Boxes C and D as nucleolar localization sequences for Box C/D snoRNAs. U14 snoRNA is shown to be required for 18S rRNA production in vertebrates.     

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.     

Box C/D snoRNP catalysed methylation is aided by additional pre-rRNA base-pairing

2'-O-methylation of eukaryotic ribosomal RNA (r)RNA, essential for ribosome function, is catalysed by box C/D small nucleolar (sno)RNPs. The RNA components of these complexes (snoRNAs) contain one or two guide sequences, which, through base-pairing, select the rRNA modification site. Adjacent to the guide sequences are protein-binding sites (the C/D or C'/D' motifs). Analysis of >2000 yeast box C/D snoRNAs identified additional conserved sequences in many snoRNAs that are complementary to regions adjacent to the rRNA methylation site. This 'extra base-pairing' was also found in many human box C/D snoRNAs and can stimulate methylation by up to five-fold. Sequence analysis, combined with RNA-protein crosslinking in Saccharomyces cerevisiae, identified highly divergent box C'/D' motifs that are bound by snoRNP proteins. In vivo rRNA methylation assays showed these to be active. Our data suggest roles for non-catalytic subunits (Nop56 and Nop58) in rRNA binding and support an asymmetric model for box C/D snoRNP organization. The study provides novel insights into the extent of the snoRNA-rRNA interactions required for efficient methylation and the structural organization of the snoRNPs.     

Location of 2(')-O-methyl nucleotides in 26S rRNA and methylation guide snoRNAs in Caenorhabditis elegans

Many nucleotides in rRNAs are modified. We devised a method to locate 2(')-O-methyl nucleotide residues using a conventional DNA sequencer. We found 38 2(')-O-methyl nucleotides in the 26S rRNA of Caenorhabditis elegans using this method. Fourteen of the 38 residues are conserved in both human and yeast rRNAs and 14 residues are conserved in either human or yeast rRNA. The remaining 10 nucleotides are uniquely methylated in C. elegans 26S rRNA. We searched the C. elegans genomic sequence for small nucleolar RNAs (snoRNAs), which guide the methylation of ribose residues, and predicted 18 snoRNA sequences that are expected to guide the methylation of some of these nucleotide residues.     

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.     

Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes

Ribose methylation is a prevalent type of nucleotide modification in rRNA. Eukaryotic rRNAs display a complex pattern of ribose methylations, amounting to 55 in yeast Saccharomyces cerevisiae and about 100 in vertebrates. Ribose methylations of eukaryotic rRNAs are each guided by a cognate small RNA, belonging to the family of box C/D antisense snoRNAs, through transient formation of a specific base-pairing at the rRNA modification site. In prokaryotes, the pattern of rRNA ribose methylations has been fully characterized in a single species so far, Escherichia coli, which contains only four ribose methylated rRNA nucleotides. However, the hyperthermophile archaeon Sulfolobus solfataricus contains, like eukaryotes, a large number of (yet unmapped) rRNA ribose methylations and homologs of eukaryotic box C/D small nucleolar ribonuclear proteins have been identified in archaeal genomes. We have therefore searched archaeal genomes for potential homologs of eukaryotic methylation guide small nucleolar RNAs, by combining searches for structured motifs with homology searches. We have identified a family of 46 small RNAs, conserved in the genomes of three hyperthermophile Pyrococcus species, which we have experimentally characterized in Pyrococcus abyssi. The Pyrococcus small RNAs, the first reported homologs of methylation guide small nucleolar RNAs in organisms devoid of a nucleus, appear as a paradigm of minimalist box C/D antisense RNAs. They differ from their eukaryotic homologs by their outstanding structural homogeneity, extended consensus box motifs and the quasi-systematic presence of two (instead of one) rRNA antisense elements. Remarkably, for each small RNA the two antisense elements always match rRNA sequences close to each other in rRNA structure, suggesting an important role in rRNA folding. Only a few of the predicted P. abyssi rRNA ribose methylations have been detected so far. Further analysis of these archaeal small RNAs could provide new insights into the origin and functions of methylation guide small nucleolar RNAs and illuminate the still elusive role of rRNA ribose methylations.     

Purified box C/D snoRNPs are able to reproduce site-specific 2'-O-methylation of target RNA in vitro

Small nucleolar RNAs (snoRNAs) are associated in ribonucleoprotein particles localized to the nucleolus (snoRNPs). Most of the members of the box C/D family function in directing site-specific 2'-O-methylation of substrate RNAs. Although the selection of the target nucleotide requires the antisense element and the conserved box D or D' of the snoRNA, the methyltransferase activity is supposed to reside in one of the protein components. Through protein tagging of a snoRNP-specific factor, we purified to homogeneity box C/D snoRNPs from the yeast Saccharomyces cerevisiae. Mass spectrometric analysis demonstrated the presence of Nop1p, Nop58p, Nop56p, and Snu13p as integral components of the particle. We show that purified snoRNPs are able to reproduce the site-specific methylation pattern on target RNA and that the predicted S-adenosyl-L-methionine-binding region of Nop1p is responsible for the catalytic activity.     

A computational screen for C/D box snoRNAs in the human genomic region associated with Prader-Willi and Angelman syndromes

Small nucleolar RNAs (snoRNAs) play a significant role in Prader-Willi Syndrome (PWS) and Angelman Syndrome (AS), which are genomic disorders resulting from deletions in the human chromosomal region 15q11–q13. To identify snoRNAs in the region, our computational study employs key motif features of C/D box snoRNAs and introduces a complementary RNA–RNA hybridization test. We identify three previously unknown methylation guide snoRNAs targeting ribosomal 18S and 28S RNAs, and two snoRNAs targeting serotonin receptor 2C mRNA. We show that the three snoRNA candidates likely possess methylation strands complementary to, and form stable complexes with, human ribosomal RNAs. Our screen also identifies 8 other snoRNA candidates that do not pass the rRNA-complementarity and/or hybridization tests. Two of these candidates have extensive sequence similarity to HBII-52, a snoRNA that regulates the alternative splicing of serotonin receptor 2C mRNA. Six out of our eleven candidate snoRNAs are also predicted by other existing methods.     

RNomics: an experimental approach that identifies 201 candidates for novel, small, non-messenger RNAs in mouse

In mouse brain cDNA libraries generated from small RNA molecules we have identified a total of 201 different expressed RNA sequences potentially encoding novel small non-messenger RNA species (snmRNAs). Based on sequence and structural motifs, 113 of these RNAs can be assigned to the C/D box or H/ACA box subclass of small nucleolar RNAs (snoRNAs), known as guide RNAs for rRNA. While 30 RNAs represent mouse homologues of previously identified human C/D or H/ACA snoRNAs, 83 correspond to entirely novel snoRNAS: Among these, for the first time, we identified four C/D box snoRNAs and four H/ACA box snoRNAs predicted to direct modifications within U2, U4 or U6 small nuclear RNAs (snRNAs). Furthermore, 25 snoRNAs from either class lacked antisense elements for rRNAs or snRNAS: Therefore, additional snoRNA targets have to be considered. Surprisingly, six C/D box snoRNAs and one H/ACA box snoRNA were expressed exclusively in brain. Of the 88 RNAs not belonging to either snoRNA subclass, at least 26 are probably derived from truncated heterogeneous nuclear RNAs (hnRNAs) or mRNAS: Short interspersed repetitive elements (SINEs) are located on five RNA sequences and may represent rare examples of transcribed SINES: The remaining RNA species could not as yet be assigned either to any snmRNA class or to a part of a larger hnRNA/mRNA. It is likely that at least some of the latter will represent novel, unclassified snmRNAS:     

Nuclear retention elements of U3 small nucleolar RNA

The processing and methylation of precursor rRNA is mediated by the box C/D small nucleolar RNAs (snoRNAs). These snoRNAs differ from most cellular RNAs in that they are not exported to the cytoplasm. Instead, these RNAs are actively retained in the nucleus where they assemble with proteins into mature small nucleolar ribonucleoprotein particles and are targeted to their intranuclear site of action, the nucleolus. In this study, we have identified the cis-acting sequences responsible for the nuclear retention of U3 box C/D snoRNA by analyzing the nucleocytoplasmic distributions of an extensive panel of U3 RNA variants after injection of the RNAs into Xenopus oocyte nuclei. Our data indicate the importance of two conserved sequence motifs in retaining U3 RNA in the nucleus. The first motif is comprised of the conserved box C' and box D sequences that characterize the box C/D family. The second motif contains conserved box sequences B and C. Either motif is sufficient for nuclear retention, but disruption of both motifs leads to mislocalization of the RNAs to the cytoplasm. Variant RNAs that are not retained also lack 5' cap hypermethylation and fail to associate with fibrillarin. Furthermore, our results indicate that nuclear retention of U3 RNA does not simply reflect its nucleolar localization. A fragment of U3 containing the box B/C motif is not localized to nucleoli but retained in coiled bodies. Thus, nuclear retention and nucleolar localization are distinct processes with differing sequence requirements.