Processing of intron-encoded box C/D small nucleolar RNAs lacking a 5',3'-terminal stem structure

The C and D box-containing (box C/D) small nucleolar RNAs (snoRNAs) function in the nucleolytic processing and 2'-O-methylation of precursor rRNA. In vertebrates, most box C/D snoRNAs are processed from debranched pre-mRNA introns by exonucleolytic activities. Elements directing accurate snoRNA excision are located within the snoRNA itself; they comprise the conserved C and D boxes and an adjoining 5',3'-terminal stem. Although the terminal stem has been demonstrated to be essential for snoRNA accumulation, many snoRNAs lack a terminal helix. To identify the cis-acting elements supporting the accumulation of intron-encoded box C/D snoRNAs devoid of a terminal stem, we have investigated the in vivo processing of the human U46 snoRNA and an artificial snoRNA from the human beta-globin pre-mRNA. We demonstrate that internal and/or external stem structures located within the snoRNA or in the intronic flanking sequences support the accumulation of mammalian box C/D snoRNAs lacking a canonical terminal stem. In the intronic precursor RNA, transiently formed external and/or stable internal base-pairing interactions fold the C and D boxes together and therefore facilitate the binding of snoRNP proteins. Since the external intronic stems are degraded during snoRNA processing, we propose that the C and D boxes alone can provide metabolic stability for the mature snoRNA.     

The SNORD115 (H/MBII-52) and SNORD116 (H/MBII-85) gene clusters at the imprinted Prader-Willi locus generate canonical box C/D snoRNAs

The imprinted Snurf-Snrpn chromosomal domain contains two large arrays of tandemly repeated, paternally expressed box C/D small-nucleolar RNA (snoRNA) genes: the SNORD115 (H/MBII-52) and SNORD116 (H/MBII-85) gene clusters believed to play key roles in the fine-tuning of serotonin receptor (5-HT2C) pre-mRNA processing and in the etiology of the Prader-Willi Syndrome (PWS), respectively. SNORD115 and SNORD116 were recently proposed to undergo significant conversion into shorter RNA species, the so-called psnoRNAs. Here, we provide evidence that argues against the existence of abundant psnoRNAs in human or mouse brain. Instead, we characterize a previously unsuspected low-abundance, fibrillarin-associated SNORD115-derived smaller RNA species. Based on these findings, we strongly recommend that PWS-encoded SNORD115 and SNORD116 be considered as bona fide box C/D snoRNAs.     

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.     

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:     

Processing of the intron-encoded U16 and U18 snoRNAs: the conserved C and D boxes control both the processing reaction and the stability of the mature snoRNA.

A novel class of small nucleolar RNAs (snoRNAs), encoded in introns of protein coding genes and originating from processing of their precursor molecules, has recently been described. The L1 ribosomal protein (r-protein) gene of Xenopus laevis and its human homologue contain two snoRNAs, U16 and U18. It has been shown that these snoRNAs are excised from their intron precursors by endonucleolytic cleavage and that their processing is alternative to splicing. Two sequences, internal to the snoRNA coding region, have been identified as indispensable for processing the conserved boxes C and D. Competition experiments have shown that these sequences interact with diffusible factors which can bind both the pre-mRNA and the mature U16 snoRNA. Fibrillarin, which is known to associate with complexes formed on C and D boxes of other snoRNAs, is found in association with mature U16 RNA, as well as with its precursor molecules. This fact suggests that the complex formed on the pre-mRNA remains bound to U16 throughout all the processing steps. We also show that the complex formed on the C and D boxes is necessary to stabilize mature snoRNA.     

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.     

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.     

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。     

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

The review considers small nucleolar RNAs (snoRNAs), an abundant group of non-protein-coding RNAs. In association with proteins, snoRNAs determine the two most common nucleotide modifications in rRNA and some other cell RNAs: 2′-O-methylation of ribose and pseudouridylation. In addition, snoRNAs are involved in pre-mRNA cleavage and the telomerase function. Almost all snoRNAs fall into two families, C/D and H/ACA, distinguished by conserved sequence boxes. Although the proteins of C/D and H/ACA snoRNPs have homologous regions, these snoRNPs are assembled differently. The RNA components of RNases P and MRP are also classed with snoRNAs. Another problem considered is the structure and function of small RNAs from Cajal bodies (small organelles associated with the nucleoli), which are similar to snoRNAs.     

Direct cloning of double-stranded RNAs from RNase protection analysis reveals processing patterns of C/D box snoRNAs and provides evidence for widespread antisense transcript expression

We describe a new method that allows cloning of double-stranded RNAs (dsRNAs) that are generated in RNase protection experiments. We demonstrate that the mouse C/D box snoRNA MBII-85 (SNORD116) is processed into at least five shorter RNAs using processing sites near known functional elements of C/D box snoRNAs. Surprisingly, the majority of cloned RNAs from RNase protection experiments were derived from endogenous cellular RNA, indicating widespread antisense expression. The cloned dsRNAs could be mapped to genome areas that show RNA expression on both DNA strands and partially overlapped with experimentally determined argonaute-binding sites. The data suggest a conserved processing pattern for some C/D box snoRNAs and abundant expression of longer, non-coding RNAs in the cell that can potentially form dsRNAs.     

核仁小RNA源性小RNA

最新研究结果表明,一些与RNA介导基因沉默相关的小RNA由核仁小RNA(small nucleolar RNA,snoRNA)加工产生,这种小RNA被称为核仁小RNA源性小RNA(snoRNA derived small RNA,sdRNA)。sdRNA现象分布物种广;涉及的snoRNA种类全,数量多;产生的小RNA分子大小不一、数量、种类多。表明这种小RNA在生物中存在着广泛的普遍性。sdRNA的发现拓展了snoRNA的功能,揭示了snoRNA与RNA介导的基因沉默之间的紧密关系,增强了snoRNA在RNA调控网络中的重要性,并为进一步研究RNA调控网络开启了一扇门。     

Guided tours: from precursor snoRNA to functional snoRNP.

Small nucleolar RNAs (snoRNAs) use base pairing to guide modification of conserved nucleotides in functionally important regions of ribosomal RNA. The box C/D snoRNAs direct 2'-O-methylation and the box H/ACA snoRNAs direct pseudouridylation. Each snoRNA interacts with proteins, many of them newly identified. Progress in understanding how snoRNA sequences are stored within genomes, liberated from precursor molecules and targeted to the nucleolus has begun to elucidate each step in the biogenesis of these critical contributors to ribosome formation.