Methodological obstacles in knocking down small noncoding RNAs

In the recent past, several thousand noncoding RNA (ncRNA) genes have been predicted within eukaryal genomes. However, for their functional analysis only a few high-throughput methods are currently available to knock down selected ncRNA species, such as microRNAs, which are targeted by antisense probes, termed antagomirs. We thus compared the efficiencies of four knockdown strategies, previously mainly employed for the analysis of protein-coding genes, to study the function of ncRNAs, in particular, small nucleolar RNAs (snoRNAs). Thereby, the class of snoRNAs represents one of the most abundant ncRNA species. The majority of snoRNAs has been shown to mediate nucleotide modifications by targeting ribosomal RNAs (rRNAs) through complementary antisense elements. However, some snoRNAs, termed “orphan snoRNAs,” lack telltale complementarities to rRNAs and thus their function remains elusive. We therefore applied RNA interference (RNAi), locked nucleic acid (LNA), or peptide nucleic acid antisense approaches, as well as a ribozyme-based strategy to knock down a snoRNA. As a proof of principle, we targeted the canonical U81 snoRNA, which has been shown to mediate modification of nucleotide A₃₉₁ within eukaryal 28S rRNA. Our results demonstrate that while RNAi is an unsuitable tool for snoRNA knockdown, a ribozyme-based strategy, as well as an LNA-antisense oligonucleotide approach, resulted in a decrease of U81 snoRNA expression levels up to 60%. However, no concomitant decrease in enzymatic activity of U81 snoRNA was observed, indicating that improvement of more efficient knockdown techniques for ncRNAs will be required in the future.     

Experimental RNomics and genomic comparative analysis reveal a large group of species-specific small non-message RNAs in the silkworm Bombyx mori

Accumulating evidences show that small non-protein coding RNAs (ncRNAs) play important roles in development, stress response and other cellular processes. The silkworm is an important model for studies on insect genetics and control of lepidopterous pests. Here, we have performed the first systematic identification and analysis of intermediate size ncRNAs (50-500 nt) in the silkworm. We identified 189 novel ncRNAs, including 141 snoRNAs, six snRNAs, three tRNAs, one SRP and 38 unclassified ncRNAs. Forty ncRNAs showed significantly altered expression during silkworm development or across specific stage transitions. Genomic comparisons revealed that 123 of these ncRNAs are potentially silkworm-specific. Analysis of the genomic organization of the ncRNA loci showed that 32.62% of the novel snoRNA loci are intergenic, and that all the intronic snoRNAs follow the pattern of one-snoRNA-per-intron. Target site analysis predicted a total of 95 2'-O-methylation and pseudouridylation modification sites of rRNAs, snRNAs and tRNAs. Together, these findings provide new clues for future functional study of ncRNA during insect development and evolution.     

Plant snoRNAs: functional evolution and new modes of gene expression

Small nucleolar RNAs (snoRNAs) are a well-characterized family of non-coding RNAs whose main function is rRNA modification. The diversity and complexity of this gene family continues to expand with the discovery of snoRNAs with non-rRNA or unknown targets. Plants contain more snoRNAs than other eukaryotes and have developed novel expression and processing strategies. The increased number of modifications, which will influence ribosome function, and the novel modes of expression might reflect the environmental conditions to which plants are exposed. Polyploidy and chromosomal rearrangements have generated multiple copies of snoRNA genes, allowing the generation of new snoRNAs for selection. The large snoRNA family in plants is an ideal model for investigation of mechanisms of evolution of gene families in plants.     

The expanding snoRNA world

In eukaryotes, the site-specific formation of the two prevalent types of rRNA modified nucleotides, 2'-O-methylated nucleotides and pseudouridines, is directed by two large families of snoRNAs. These are termed box C/D and H/ACA snoRNAs, respectively, and exert their function through the formation of a canonical guide RNA duplex at the modification site. In each family, one snoRNA acts as a guide for one, or at most two modifications, through a single, or a pair of appropriate antisense elements. The two guide families now appear much larger than anticipated and their role not restricted to ribosome synthesis only. This is reflected by the recent detection of guides that can target other cellular RNAs, including snRNAs, tRNAs and possibly even mRNAs, and by the identification of scores of tissue-specific specimens in mammals. Recent characterization of homologs of eukaryotic modification guide snoRNAs in Archaea reveals the ancient origin of these non-coding RNA families and offers new perspectives as to their range of function.     

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.     

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.     

Systematic identification and characterization of chicken (Gallus gallus) ncRNAs

Recent studies have demonstrated that non-coding RNAs (ncRNAs) play important roles during development and evolution. Chicken, the first genome-sequenced non-mammalian amniote, possesses unique features for developmental and evolutionary studies. However, apart from microRNAs, information on chicken ncRNAs has mainly been obtained from computational predictions without experimental validation. In the present study, we performed a systematic identification of intermediate size ncRNAs (50–500 nt) by ncRNA library construction and identified 125 chicken ncRNAs. Importantly, through the bioinformatics and expression analysis, we found the chicken ncRNAs has several novel features: (i) comparative genomic analysis against 18 sequenced vertebrate genomes revealed that the majority of the newly identified ncRNA candidates is not conserved and most are potentially bird/chicken specific, suggesting that ncRNAs play roles in lineage/species specification during evolution. (ii) The expression pattern analysis of intronic snoRNAs and their host genes suggested the coordinated expression between snoRNAs and their host genes. (iii) Several spatio-temporal specific expression patterns suggest involvement of ncRNAs in tissue development. Together, these findings provide new clues for future functional study of ncRNAs during development and evolution.     

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.     

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:     

Identification of 13 novel human modification guide RNAs

Members of the two expanding RNA subclasses termed C/D and H/ACA RNAs guide the 2'-O-methylations and pseudouridylations, respectively, of rRNA and spliceosomal RNAs (snRNAs). Here, we report on the identification of 13 novel human intron-encoded small RNAs (U94-U106) belonging to the two subclasses of modification guides. Seven of them are predicted to direct 2'-O-methylations in rRNA or snRNAs, while the remainder represent novel orphan RNA modification guides. From these, U100, which is exclusively detected in Cajal bodies (CBs), is predicted to direct modification of a U6 snRNA uridine, U(9), which to date has not been found to be pseudouridylated. Hence, within CBs, U100 might function in the folding pathway or other aspects of U6 snRNA metabolism rather than acting as a pseudouridylation guide. U106 C/D snoRNA might also possess an RNA chaperone activity only since its two conserved antisense elements match two rRNA sequences devoid of methylated nucleotides and located remarkably close to each other within the 18S rRNA secondary structure. Finally, we have identified a retrogene for U99 snoRNA located within an intron of the Siat5 gene, supporting the notion that retro-transposition events might have played a substantial role in the mobility and diversification of snoRNA genes during evolution.     

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.     

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.