Plant U13 orthologues and orphan snoRNAs identified by RNomics of RNA from Arabidopsis nucleoli

Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs whose main function in eukaryotes is to guide the modification of nucleotides in ribosomal and spliceosomal small nuclear RNAs, respectively. Full-length sequences of Arabidopsis snoRNAs and scaRNAs have been obtained from cDNA libraries of capped and uncapped small RNAs using RNA from isolated nucleoli from Arabidopsis cell cultures. We have identified 31 novel snoRNA genes (9 box C/D and 22 box H/ACA) and 15 new variants of previously described snoRNAs. Three related capped snoRNAs with a distinct gene organization and structure were identified as orthologues of animal U13snoRNAs. In addition, eight of the novel genes had no complementarity to rRNAs or snRNAs and are therefore putative orphan snoRNAs potentially reflecting wider functions for these RNAs. The nucleolar localization of a number of the snoRNAs and the localization to nuclear bodies of two putative scaRNAs was confirmed by in situ hybridization. The majority of the novel snoRNA genes were found in new gene clusters or as part of previously described clusters. These results expand the repertoire of Arabidopsis snoRNAs to 188 snoRNA genes with 294 gene variants.     

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.     

Different expression strategy: multiple intronic gene clusters of box H/ACA snoRNA in Drosophila melanogaster

The high degree of rRNA pseudouridylation in Drosophila melanogaster provides a good model for studying the genomic organization, structural and functional diversity of box H/ACA small nucleolar RNAs (snoRNAs). Accounting for both conserved sequence motifs and secondary structures, we have developed a computer-assisted method for box H/ACA snoRNA searching. Ten snoRNA clusters containing 42 box H/ACA snoRNAs were identified from D.melanogaster. Strikingly, they are located in the introns of eight protein-coding genes. In contrast to the mode of one snoRNA per intron so far observed in all animals, our results demonstrate for the first time a novel polycistronic organization that implies a different expression strategy for a box H/ACA snoRNA gene when compared to box C/D snoRNAs in D.melanogaster. Mutiple isoforms of the box H/ACA snoRNAs, from which most clusters are made up, were observed in D.melanogaster. The degree of sequence similarity between the isoforms varies from 99% to 70%, implying duplication events in different periods and a trend of enlarging the intronic snoRNA clusters. The variation in the functional elements of the isoforms could lead to partial alternation of snoRNA's function in loss or gain of rRNA complementary sequences and probably contributes to the great diversity of rRNA pseudouridylation in D.melanogaster.     

Genomewide analysis of box C/D and box H/ACA snoRNAs in Chlamydomonas reinhardtii reveals an extensive organization into intronic gene clusters

Chlamydomonas reinhardtii is a unicellular green alga, the lineage of which diverged from that of land plants >1 billion years ago. Using the powerful small nucleolar RNA (snoRNA) mining platform to screen the C. reinhardtii genome, we identified 322 snoRNA genes grouped into 118 families. The 74 box C/D families can potentially guide methylation at 96 sites of ribosomal RNAs (rRNAs) and snRNAs, and the 44 box H/ACA families can potentially guide pseudouridylation at 62 sites. Remarkably, 242 of the snoRNA genes are arranged into 76 clusters, of which 77% consist of homologous genes produced by small local tandem duplications. At least 70 snoRNA gene clusters are found within introns of protein-coding genes. Although not exhaustive, this analysis reveals that C. reinhardtii has the highest number of intronic snoRNA gene clusters among eukaryotes. The prevalence of intronic snoRNA gene clusters in C. reinhardtii is similar to that of rice but in contrast with the one-snoRNA-per-intron organization of vertebrates and fungi and with that of Arabidopsis thaliana in which only a few intronic snoRNA gene clusters were identified. This analysis of C. reinhardtii snoRNA gene organization shows the functional importance of introns in a single-celled organism and provides evolutionary insight into the origin of intron-encoded RNAs in the plant lineage.     

The genes for small nucleolar RNAs in Trypanosoma brucei are organized in clusters and are transcribed as a polycistronic RNA

Because the organization of snoRNA genes in vertebrates, plants and yeast is diverse, we investigated the organization of snoRNA genes in a distantly related organism, Trypanosoma brucei. We have characterized the second example of a snoRNA gene cluster that is tandemly repeated in the T.brucei genome. The genes encoding the box C/D snoRNAs TBR12, TBR6, TBR4 and TBR2 make up the cluster. In a genomic organization unique to trypanosomes, there are at least four clusters of these four snoRNA genes tandemly repeated in the T.brucei genome. We show for the first time that the genes encoding snoRNAs in both this cluster and the SLA cluster are transcribed in an unusual way as a polycistronic RNA.     

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.     

Probing RNA in vivo with methylation guide small nucleolar RNAs

Most box C/D small nucleolar RNAs (snoRNAs) direct the formation of 2'-O-methylated nucleotides in ribosomal RNA and, apparently, other RNAs present in the nucleolar complex. Sites to be modified are selected by a long (>10-nt) antisense guide sequence in the snoRNA and a distance measurement from a box D or D' element that follows the snoRNA guide sequence. Modification of the substrate occurs in the region of complementarity, at a position five nucleotides upstream from box D/D'. Methylation can be targeted to novel sites by expressing a snoRNA with a new guide sequence. In some cases methylation impairs the growth rate of the cell, indicating that a functionally important nucleotide has been altered. With a view to harnessing snoRNA-directed methylation for functional mapping, we have developed a method for constructing libraries of snoRNA genes that, in principle, can introduce methylation point mutations into any rRNA segment of interest. The strategy and procedures are described here, and preliminary results are presented that show the feasibility of using this technology to probe a region of the yeast large subunit rRNA that includes the core of the peptidyltransferase center.     

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.     

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.     

Elucidating the role of C/D snoRNA in rRNA processing and modification in Trypanosoma brucei

Most eukaryotic C/D small nucleolar RNAs (snoRNAs) guide 2′-O methylation (Nm) on rRNA and are also involved in rRNA processing. The four core proteins that bind C/D snoRNA in Trypanosoma brucei are fibrillarin (NOP1), NOP56, NOP58, and SNU13. Silencing of NOP1 by RNA interference identified rRNA-processing and modification defects that caused lethality. Systematic mapping of 2′-O-methyls on rRNA revealed the existence of hypermethylation at certain positions of the rRNA in the bloodstream form of the parasites, suggesting that this modification may assist the parasites in coping with the major temperature changes during cycling between their insect and mammalian hosts. The rRNA-processing defects of NOP1-depleted cells suggest the involvement of C/D snoRNA in trypanosome-specific rRNA-processing events to generate the small rRNA fragments. MRP RNA, which is involved in rRNA processing, was identified in this study in one of the snoRNA gene clusters, suggesting that trypanosomes utilize a combination of unique C/D snoRNAs and conserved snoRNAs for rRNA processing.     

Mining small RNA sequencing data: a new approach to identify small nucleolar RNAs in Arabidopsis

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that direct 2′-O-methylation or pseudouridylation on ribosomal RNAs or spliceosomal small nuclear RNAs. These modifications are needed to modulate the activity of ribosomes and spliceosomes. A comprehensive repertoire of snoRNAs is needed to expand the knowledge of these modifications. The sequences corresponding to snoRNAs in 18–26-nt small RNA sequencing data have been rarely explored and remain as a hidden treasure for snoRNA annotation. Here, we showed the enrichment of small RNAs at Arabidopsis snoRNA termini and developed a computational approach to identify snoRNAs on the basis of this characteristic. The approach successfully uncovered the full-length sequences of 144 known Arabidopsis snoRNA genes, including some snoRNAs with improved 5′- or 3′-end annotation. In addition, we identified 27 and 17 candidates for novel box C/D and box H/ACA snoRNAs, respectively. Northern blot analysis and sequencing data from parallel analysis of RNA ends confirmed the expression and the termini of the newly predicted snoRNAs. Our study especially expanded on the current knowledge of box H/ACA snoRNAs and snoRNA species targeting snRNAs. In this study, we demonstrated that the use of small RNA sequencing data can increase the complexity and the accuracy of snoRNA annotation.