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.     

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:     

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.     

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。     

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.     

18S rRNA processing requires base pairings of snR30 H/ACA snoRNA to eukaryote‐specific 18S sequences

The H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse class of non‐coding RNAs. Many H/ACA RNAs direct pseudouridylation of rRNAs and snRNAs, while members of the rapidly growing group of ‘orphan’ H/ACA RNAs participate in pre‐rRNA processing, telomere synthesis and probably, in other nuclear processes. The yeast snR30 ‘orphan’ H/ACA snoRNA has long been known to function in the nucleolytic processing of 18S rRNA, but its molecular role remained unknown. Here, we provide biochemical and genetic evidence demonstrating that during pre‐rRNA processing, two evolutionarily conserved sequence elements in the 3′‐hairpin of snR30 base‐pair with short pre‐rRNA sequences located in the eukaryote‐specific internal region of 18S rRNA. The newly discovered snR30‐18S base‐pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs. We also demonstrate that besides the 18S recognition motifs, the distal part of the 3′‐hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein‐binding site.     

A search for H/ACA snoRNAs in yeast using MFE secondary structure prediction

MOTIVATION: Noncoding RNA genes produce functional RNA molecules rather than coding for proteins. One such family is the H/ACA snoRNAs. Unlike the related C/D snoRNAs these have resisted automated detection to date. RESULTS: We develop an algorithm to screen the yeast genome for novel H/ACA snoRNAs. To achieve this, we introduce some new methods for facilitating the search for noncoding RNAs in genomic sequences which are based on properties of predicted minimum free-energy (MFE) secondary structures. The algorithm has been implemented and can be generalized to enable screening of other eukaryote genomes. We find that use of primary sequence alone is insufficient for identifying novel H/ACA snoRNAs. Only the use of secondary structure filters reduces the number of candidates to a manageable size. From genomic context, we identify three strong H/ACA snoRNA candidates. These together with a further 47 candidates obtained by our analysis are being experimentally screened.