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.     

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:     

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.     

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.     

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.     

The snoGloBe interaction predictor reveals a broad spectrum of C/D snoRNA RNA targets

Box C/D small nucleolar RNAs (snoRNAs) are a conserved class of RNA known for their role in guiding ribosomal RNA 2′-O-ribose methylation. Recently, C/D snoRNAs were also implicated in regulating the expression of non-ribosomal genes through different modes of binding. Large scale RNA–RNA interaction datasets detect many snoRNAs binding messenger RNA, but are limited by specific experimental conditions. To enable a more comprehensive study of C/D snoRNA interactions, we created snoGloBe, a human C/D snoRNA interaction predictor based on a gradient boosting classifier. SnoGloBe considers the target type, position and sequence of the interactions, enabling it to outperform existing predictors. Interestingly, for specific snoRNAs, snoGloBe identifies strong enrichment of interactions near gene expression regulatory elements including splice sites. Abundance and splicing of predicted targets were altered upon the knockdown of their associated snoRNA. Strikingly, the predicted snoRNA interactions often overlap with the binding sites of functionally related RNA binding proteins, reinforcing their role in gene expression regulation. SnoGloBe is also an excellent tool for discovering viral RNA targets, as shown by its capacity to identify snoRNAs targeting the heavily methylated SARS-CoV-2 RNA. Overall, snoGloBe is capable of identifying experimentally validated binding sites and predicting novel sites with shared regulatory function.     

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.     

Distribution of U3 small nucleolar RNA and fibrillarin during early embryogenesis in Caenorhabditis elegans

U3 small nucleolar RNA (snoRNA) is one of the members of the box C/D class of snoRNA and is essential for ribosomal RNA (rRNA) processing to generate 18S rRNA in the nucleolus. Although U3 snoRNA is abundant, and is well conserved from yeast to mammals, the genes encoding U3 snoRNA in C. elegans have long remained unidentified. A recent RNomics study in C. elegans predicted five distinct U3 snoRNA genes. However, characterization of these candidates for U3 snoRNA has yet to be performed. In this study, we isolated and characterized four candidate RNAs for U3 snoRNA from the immunoprecipitated RNAs of C. elegans using an antibody against the 2,2,7-trimethylguanosine (TMG) cap. The sequences were identical to the predicted U3 sequences in the RNomics study. Here, we show the several lines of evidence that the isolated RNAs are the true U3 snoRNAs of C. elegans. Moreover, we report the novel expression pattern of U3 snoRNA and fibrillarin, which is an essential component of U3 small nucleolar ribonucleoprotein complex, during early embryo development of C. elegans. To our knowledge, this is the first observation of the inconsistent localization U3 snoRNA and fibrillarin during early embryogenesis, providing novel insight into the mechanisms of nucleologenesis and ribosome production during early embryogenesis.     

AtNUFIP, an essential protein for plant development, reveals the impact of snoRNA gene organisation on the assembly of snoRNPs and rRNA methylation in Arabidopsis thaliana

In all eukaryotes, C/D small nucleolar ribonucleoproteins (C/D snoRNPs) are essential for methylation and processing of ribosomal RNAs. They consist of a box C/D small nucleolar RNA (C/D snoRNA) associated with four highly conserved nucleolar proteins. Recent data in HeLa cells and yeast have revealed that assembly of these snoRNPs is directed by NUFIP protein and other auxiliary factors. Nevertheless, the precise function and biological importance of NUFIP and the other assembly factors remains unknown. In plants, few studies have focused on RNA methylation and snoRNP biogenesis. Here, we identify and characterise the AtNUFIP gene that directs assembly of C/D snoRNP. To elucidate the function of AtNUFIP in planta, we characterized atnufip mutants. These mutants are viable but have severe developmental phenotypes. Northern blot analysis of snoRNA accumulation in atnufip mutants revealed a specific degradation of C/D snoRNAs and this situation is correlated with a reduction in rRNA methylation. Remarkably, the impact of AtNUFIP depends on the structure of snoRNA genes: it is essential for the accumulation of those C/D snoRNAs encoded by polycistronic genes, but not by monocistronic or tsnoRNA genes. We propose that AtNUFIP controls the kinetics of C/D snoRNP assembly on nascent precursors to overcome snoRNA degradation of aberrant RNPs. Finally, we show that AtNUFIP has broader RNP targets, controlling the accumulation of scaRNAs that direct methylation of spliceosomal snRNA in Cajal bodies.     

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。     

Analysis of Snu13p mutations reveals differential interactions with the U4 snRNA and U3 snoRNA

Pre-mRNA splicing is executed by the spliceosome, a complex of small nuclear RNAs (snRNAs) and numerous proteins. One such protein, 15.5K/Snu13p, is associated with the spliceosomal U4/U6.U5 tri-snRNP and box C/D small nucleolar ribonucleoprotein particles (snoRNPs), which act during preribosomal RNA (rRNA) processing. As such, it is the first splicing factor to be identified in two functionally distinct particles. 15.5K binds to an internal helix-bulge-helix (K-turn) structure in the U4 snRNA and two such structures in the U3 snoRNA. Previous work has concentrated on the structural basis of the interaction of 15.5K with the RNAs and has been carried out in vitro. Here we present a functional analysis of Snu13p in vivo, using a galactose inducible SNU13 strain to investigate the basis of three lethal mutations in Saccharomyces cerevisiae. Two are point mutations that map to the RNA-binding domain, and the third is a C-terminal deletion. These mutations result in accumulation of unspliced pre-mRNA, confirming a role for Snu13p in pre-mRNA splicing. In addition, these mutants also display rRNA processing defects that are variable in nature. Analysis of one mutant in the RNA-binding domain reveals a reduction in the levels of the U4 snRNA, U6 snRNA, and box C/D snoRNAs, but not H/ACA snoRNAs, supporting a role for Snu13p in accumulation and/or maintenance of specific RNAs. The mutations in the RNA-binding domain exhibit differential binding to the U4 snRNA and U3 snoRNA in vitro, suggesting that there are differences in the mode of interaction of Snu13p with these two RNAs.