Genome-wide analysis of C/D and H/ACA-like small nucleolar RNAs in Leishmania major indicates conservation among trypanosomatids in the repertoire and in their rRNA targets

Small nucleolar RNAs (snoRNAs) are a large group of noncoding RNAs that exist in eukaryotes and archaea and guide modifications such as 2'-O-ribose methylations and pseudouridylation on rRNAs and snRNAs. Recently, we described a genome-wide screening approach with Trypanosoma brucei that revealed over 90 guide RNAs. In this study, we extended this approach to analyze the repertoire of the closely related human pathogen Leishmania major. We describe 23 clusters that encode 62 C/Ds that can potentially guide 79 methylations and 37 H/ACA-like RNAs that can potentially guide 30 pseudouridylation reactions. Like T. brucei, Leishmania also contains many modifications and guide RNAs relative to its genome size. This study describes 10 H/ACAs and 14 C/Ds that were not found in T. brucei. Mapping of 2'-O-methylations in rRNA regions rich in modifications suggests the existence of trypanosomatid-specific modifications conserved in T. brucei and Leishmania. Structural features of C/D snoRNAs, such as copy number, conservation of boxes, K turns, and intragenic and extragenic base pairing, were examined to elucidate the great variation in snoRNA abundance. This study highlights the power of comparative genomics for determining conserved features of noncoding RNAs.     

Pseudouridine-guide RNAs and other Cbf5p-associated RNAs in Euglena gracilis

In eukaryotes, box H/ACA small nucleolar RNAs (snoRNAs) guide sites of pseudouridine (Psi) formation in rRNA. These snoRNAs reside in RNP complexes containing the putative Psi synthase, Cbf5p. In this study we have identified Cbf5p-associated RNAs in Euglena gracilis, an early diverging eukaryote, by immunoprecipitating Cbf5p-containing complexes from cellular extracts. We characterized one box H/ACA-like RNA which, however, does not appear to guide Psi formation in rRNA. We also identified four single Psi-guide box AGA RNAs. We determined target sites for these putative Psi-guide RNAs and confirmed that the predicted Psi modifications do, in fact, occur at these positions in Euglena rRNA. The Cbf5p-associated snoRNAs appear to be encoded by multicopy genes, some of which are clustered in the genome together with methylation-guide snoRNA genes. These modification-guide snoRNAs and snoRNA genes are the first ones to be reported in euglenid protists, the evolutionary sister group to the kinetoplastid protozoa. Unexpectedly, we also found and have partially characterized a selenocysteine tRNA homolog in the anti-Cbf5p-immunoprecipitated sample.     

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.     

Identification of 66 box C/D snoRNAs in Arabidopsis thaliana: extensive gene duplications generated multiple isoforms predicting new ribosomal RNA 2'-O-methylation sites

Dozens of box C/D small nucleolar RNAs (snoRNAs) have recently been found in eukaryotes (vertebrates, yeast), ancient eukaryotes (trypanosomes) and archae, that specifically target ribosomal RNA sites for 2'-O-ribose methylation. Although early biochemical data revealed that plant rRNAs are among the most highly ribomethylated in eukaryotes, only a handful of methylation guide snoRNAs have been characterized in this kingdom. We report 66 novel box C/D snoRNAs identified by computational screening of Arabidopsis genomic sequences that are expressed in vivo from either single genes, 17 different clusters or three introns. At the structural level, many box C/D snoRNAs have dual antisense elements often matching rRNA regions close to each other on the rRNA secondary structure, which is reminiscent of their archaeal counterparts. Remarkable specimens are found that display two antisense elements having the potential to form an extended snoRNA-rRNA duplex of 23 to 30 nt, in line with the hypothetical function of box C/D snoRNAs in pre-rRNA folding or chaperoning. In contrast to other species, many Arabidopsis snoRNAs are found in multiple isoforms mainly resulting from two different mechanisms: large chromosomal duplications and small tandem duplications producing polycistronic genes. The discovery of numerous different snoRNAs, some of them arising from common ancestors, provide new insights to understand snoRNAs evolution and the birth of new rRNA methylation sites in plants and other organisms.     

The complete set of H/ACA snoRNAs that guide rRNA pseudouridylations in Saccharomyces cerevisiae

Conversion of uridines into pseudouridines (Psis) is the most frequent base modification in ribosomal RNAs (rRNAs). In eukaryotes, the pseudouridylation sites are specified by base-pairing with specific target sequences within H/ACA small nucleolar RNAs (snoRNAs). The yeast rRNAs harbor 44 Psis, but, when this work began, 15 Psis had completely unknown guide snoRNAs. This suggested that many snoRNAs remained to be discovered. To address this problem and further complete the snoRNA assignment to Psi sites, we identified the complete set of RNAs associated with the H/ACA snoRNP specific proteins Gar1p and Nhp2p by coupling TAP-tag purifications with genomic DNA microarrays experiments. Surprisingly, while we identified all the previously known H/ACA snoRNAs, we selected only three new snoRNAs. This suggested that most of the missing Psi guides were present in previously known snoRNAs but had been overlooked. We confirmed this hypothesis by systematically investigating the role of previously known, as well as of the newly identified snoRNAs, in specifying rRNA Psi sites and found all but one missing guide RNAs. During the completion of this work, another study, based on bioinformatic predictions, also reported the identification of most missing guide RNAs. Altogether, all Psi guides are now identified and we can tell that, in budding yeast, the 44 Psis are guided by 28 snoRNAs. Finally, aside from snR30, an atypical small RNA of heterogeneous length and at least one mRNA, all Gar1p and Nhp2p associated RNAs characterized by our work turned out to be snoRNAs involved in rRNA Psi specification.     

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.     

Small nucleolar RNA genes

Small nucleolar RNAs (snoRNAs) are one of the most numerous and well-studied groups of non-protein-coding RNAs. In complex with proteins, snoRNAs perform the two most common nucleotide modifications in rRNA: 2′-OH-methylation of ribose and pseudouridylation. Although the modification mechanisms and snoRNP structures are highly conserved, the snoRNA genes are surprisingly diverse in organization. In addition to genes transcribed independently, there are genes that are in introns of other genes, form clusters transcribed from a common promoter, or clusters in introns. Interestingly, one type of gene organization usually prevails in different taxa. Vertebrate snoRNAs mostly originate from introns of protein-coding genes; a small group of snoRNAs are encoded by introns of genes for noncoding RNAs.     

A modification of Representational Difference Analysis, with application to the cloning of a candidate in the Reelin signalling pathway

Background  

Ribose 2'-O-methylation, the most common nucleotide modification in mammalian rRNA, is directed by the C/D box small nucleolar RNAs (snoRNAs). Thus far, more than fifty putative human rRNA methylation guide snoRNAs have been identified. For nine of these snoRNAs, the respective ribose methylations in human 28S rRNA have been only presumptive.     

Small nucleolar RNA genes

Small nucleolar RNAs (snoRNAs) are one of the most numerous and well-studied groups of non-protein-coding RNAs. In complex with proteins, snoRNAs perform the two most common nucleotide modifications in rRNA: 2'-O-methylation of ribose and pseudouridylation. Although the modification mechanisms and shoRNA structures are highly conserved, the snoRNA genes are surprisingly diverse in organization. In addition to genes transcribed independently, there are genes that are in introns of other genes, form clusters transcribed from a common promoter, or cluster in introns. Interestingly. one type of gene organization usually prevails in different taxa. Vertebrate snoRNAs mostly originate from introns of protein-coding genes; a small group of snoRNAs are encoded by introns of genes for noncoding RNAs.     

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.     

A snoRNA that guides the two most conserved pseudouridine modifications within rRNA confers a growth advantage in yeast

Ribosomal RNAs contain a number of modified nucleotides. The most abundant nucleotide modifications found within rRNAs fall into two types: 2'-O-ribose methylations and pseudouridylations. In eukaryotes, small nucleolar guide RNAs, the snoRNAs that are the RNA components of the snoRNPs, specify the position of these modifications. The 2'-O-ribose methylations and pseudouridylations are guided by the box C/D and box H/ACA snoRNAs, respectively. The role of these modifications in rRNA remains poorly understood as no clear phenotype has yet been assigned to the absence of specific 2'-O-ribose methylations or pseudouridylations. Only very recently, a slight translation defect and perturbation of polysome profiles was reported in yeast for the absence of the Psi at position 2919 within the LSU rRNA. Here we report the identification and characterization in yeast of a novel intronic H/ACA snoRNA that we called snR191 and that guides pseudouridylation at positions 2258 and 2260 in the LSU rRNA. Most interestingly, these two modified bases are the most conserved pseudouridines from bacteria to human in rRNA. The corresponding human snoRNA is hU19. We show here that, in yeast, the presence of this snoRNA, and hence, most likely, of the conserved pseudouridines it specifies, is not essential for viability but provides a growth advantage to the cell.