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:     

RNomics in Drosophila melanogaster: identification of 66 candidates for novel non-messenger RNAs

By generating a specialised cDNA library from four different developmental stages of Drosophila melanogaster, we have identified 66 candidates for small non-messenger RNAs (snmRNAs) and have confirmed their expression by northern blot analysis. Thirteen of them were expressed at certain stages of D.melanogaster development, only. Thirty-five species belong to the class of small nucleolar RNAs (snoRNAs), divided into 15 members from the C/D subclass and 20 members from the H/ACA subclass, which mostly guide 2'-O-methylation and pseudouridylation, respectively, of rRNA and snRNAs. These also include two outstanding C/D snoRNAs, U3 and U14, both functioning as pre-rRNA chaperones. Surprisingly, the sequence of the Drosophila U14 snoRNA reflects a major change of function of this snoRNA in Diptera relative to yeast and vertebrates. Among the 22 snmRNAs lacking known sequence and structure motifs, five were located in intergenic regions, two in introns, five in untranslated regions of mRNAs, eight were derived from open reading frames, and two were transcribed opposite to an intron. Interestingly, detection of two RNA species from this group implies that certain snmRNA species are processed from alternatively spliced pre-mRNAs. Surprisingly, a few snmRNA sequences could not be found on the published D.melanogaster genome, which might suggest that more snmRNA genes (as well as mRNAs) are hidden in unsequenced regions of the genome.     

RNomics: identification and function of small,non-messenger RNAs

In the past few years, our knowledge about small non-mRNAs (snmRNAs) has grown exponentially. Approaches including computational and experimental RNomics have led to a plethora of novel snmRNAs, especially small nucleolar RNAs (snoRNAs). Members of this RNA class guide modification of ribosomal and spliceosomal RNAs. Novel targets for snoRNAs were identified such as tRNAs and potentially mRNAs, and several snoRNAs were shown to be tissue-specifically expressed. In addition, previously unknown classes of snmRNAs have been discovered. MicroRNAs and small interfering RNAs of about 21-23 nt, were shown to regulate gene expression by binding to mRNAs via antisense elements. Regulation of gene expression is exerted by degradation of mRNAs or translational regulation. snmRNAs play a variety of roles during regulation of gene expression. Moreover, the function of some snmRNAs known for decades, has been finally elucidated. Many other RNAs were identified by RNomics studies lacking known sequence and structure motifs. Future challenges in the field of RNomics include identification of the novel snmRNA's biological roles in the cell.     

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.     

动物snmRNA的系统识别与鉴定

小分子非编码RNA(snmRNA)在调控基因的转录和转录后加工、细胞分化和个体发育、遗传和表观遗传等几乎所有的重要生命活动中发挥关键作用。建立和发展snmRNA研究技术,系统地发现和注释基因组中的snmRNA基因并阐明其生物学意义是当前RNA组学的首要任务。围绕snmRNA的系统识别与鉴定等问题,该文对近年来采用实验技术和计算机预测方法发掘snmRNA所取得的主要研究成果进行综述。     

Prediction of structured non-coding RNAs in the genomes of the nematodes Caenorhabditis elegans and Caenorhabditis briggsae

We present a survey for non-coding RNAs and other structured RNA motifs in the genomes of Caenorhabditis elegans and Caenorhabditis briggsae using the RNAz program. This approach explicitly evaluates comparative sequence information to detect stabilizing selection acting on RNA secondary structure. We detect 3,672 structured RNA motifs, of which only 678 are known non-translated RNAs (ncRNAs) or clear homologs of known C. elegans ncRNAs. Most of these signals are located in introns or at a distance from known protein-coding genes. With an estimated false positive rate of about 50% and a sensitivity on the order of 50%, we estimate that the nematode genomes contain between 3,000 and 4,000 RNAs with evolutionary conserved secondary structures. Only a small fraction of these belongs to the known RNA classes, including tRNAs, snoRNAs, snRNAs, or microRNAs. A relatively small class of ncRNA candidates is associated with previously observed RNA-specific upstream elements.     

细菌全局性调控因子CsrA的结构与功能

碳存储调控因子A (carbon storage regulator, CsrA) 是一种RNA结合蛋白,在细菌的碳代谢、生物被膜形成、运动性、病原菌毒力、群体感应、环二鸟苷酸信号合成、应激感应等多种生理过程中具有重要调节功能,是全局性调控蛋白.它通过与靶标mRNA的特异结合,抑制其翻译或增强其稳定性来调控下游基因的表达,属于转录后调控因子的范畴.CsrA蛋白的表达与活性受碳存储调控(Csr)系统本身多个自主调节回路的精密控制：　一些小的非编码RNA (snmRNAs,如CsrB/C）作为拮抗因子与CsrA二聚体结合并抑制其活性;而这些snmRNAs在体内又可在CsrD的辅助下被核糖核酸内切酶E和多核苷酸磷酸化酶降解,释放CsrA的活性.当前,对于Csr系统的调节作用、调控通路与机制的研究是细菌学研究的热点,本文综述了该蛋白及Csr系统的结构、功能和作用机制的最新研究进展.     

Novel non-coding RNAs in Dictyostelium discoideum and their expression during development

The quest for non-coding RNAs (ncRNAs) in the last few years has revealed a surprisingly large number of small RNAs belonging to previously known as well as entirely novel classes. Computational and experimental approaches have uncovered new ncRNAs in all kingdoms of life. In this work, we used a shotgun cloning approach to construct full-length cDNA libraries of small RNAs from the eukaryotic model organism Dictyostelium discoideum. Interestingly, two entirely novel classes of RNAs were identified of which one is developmentally regulated. The RNAs within each class share conserved 5'- and 3'-termini that can potentially form stem structures. RNAs of both classes show predominantly cytoplasmic localization. In addition, based on conserved structure and/or sequence motifs, several of the identified ncRNAs could be divided into classes known from other organisms, e.g. 18 small nucleolar RNA candidates (17 box C/D, of which a few are developmentally regulated, and one box H/ACA). Two ncRNAs showed a high degree of similarity to the small nuclear U2 RNA and signal recognition particle RNA (SRP RNA), respectively. Furthermore, the majority of the regions upstream of the sequences encoding the isolated RNAs share conserved motifs that may constitute new promoter elements.     

Small RNA library preparation for next-generation sequencing by single ligation,extension and circularization technology

The discovery of novel small RNA classes and species has accelerated since the implementation of high-throughput sequencing technologies for the identification of small RNAs. However, as the sequence coverage increases in a cell, the expectation of finding novel small RNAs from a batch of sequencing gradually decreases. To improve the finding of novel small RNAs, an alternative small RNA library preparation method, the single ligation, extension and circularization method, has been developed which is adequate for high throughput sequencing. The procedure is faster and simpler than the more widely used procedures, and the constructed libraries are compatible with high-level multiplex analysis. The analysis of human small RNA libraries prepared by the SLEC method reported known small RNAs and novel small RNAs including 25 mirtron candidates. This study demonstrates that the method is effective in identifying known and novel small RNAs.     

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.     

Plant dicistronic tRNA-snoRNA genes: a new mode of expression of the small nucleolar RNAs processed by RNase Z

Small nucleolar RNAs (snoRNAs) guiding modifications of ribosomal RNAs and other RNAs display diverse modes of gene organization and expression depending on the eukaryotic system: in animals most are intron encoded, in yeast many are monocistronic genes and in plants most are polycistronic (independent or intronic) genes. Here we report an unprecedented organization: plant dicistronic tRNA-snoRNA genes. In Arabidopsis thaliana we identified a gene family encoding 12 novel box C/D snoRNAs (snoR43) located just downstream from tRNA(Gly) genes. We confirmed that they are transcribed, probably from the tRNA gene promoter, producing dicistronic tRNA(Gly)-snoR43 precursors. Using transgenic lines expressing a tagged tRNA-snoR43.1 gene we show that the dicistronic precursor is accurately processed to both snoR43.1 and tRNA(Gly). In addition, we show that a recombinant RNase Z, the plant tRNA 3' processing enzyme, efficiently cleaves the dicistronic precursor in vitro releasing the snoR43.1 from the tRNA(Gly). Finally, we describe a similar case in rice implicating a tRNA(Met-e) expressed in fusion with a novel C/D snoRNA, showing that this mode of snoRNA expression is found in distant plant species.     

植物一个新box H/ACA snoRNA的鉴定及功能分析

通过生物信息学方法对拟南芥基因组序列进行搜索,发现两个新的非编码小分子RNA基因,分别命名为AthsnoR206 a和AthsnoR206b。它们相距约170nt,位于蛋白质基因间隔区。MFOLD二级结构预测这两个RNA均具有典型的box H/ACAsnoRNA"发夹-铰链-发夹-尾巴"结构,符合box H/ACA snoRNA的判定标准;两个RNA分子的反义序列一致,可以判定它们为同一基因的两个拷贝。分析预测snoR206的两段反义序列分别指导拟南芥rRNA小亚基U1717位点和大亚基U2181位点的假尿嘧啶化修饰。在其它13种包括单子叶植物和双子叶植物在内的植物搜索到14个snoR206同源分子,其中12个发现于表达序列标签中,表明该snoRNA在植物中表达且广泛存在。具有双功能的snoR206在人和酵母中的部分功能同源分子分别为U70和snR32,表明其祖先分子在进化过程中存在分子重组。     

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.     

A small nucleolar guide RNA functions both in 2'-O-ribose methylation and pseudouridylation of the U5 spliceosomal RNA

In eukaryotes, two distinct classes of small nucleolar RNAs (snoRNAs), namely the fibrillarin-associated box C/D snoRNAs and the Gar1p-associated box H/ACA snoRNAs, direct the site-specific 2'-O-ribose methylation and pseudouridylation of ribosomal RNAs (rRNAs), respectively. We have identified a novel evolutionarily conserved snoRNA, called U85, which possesses the box elements of both classes of snoRNAs and associates with both fibrillarin and Gar1p. In vitro and in vivo pseudouridylation and 2'-O-methylation experiments provide evidence that the U85 snoRNA directs 2'-O-methylation of the C45 and pseudouridylation of the U46 residues in the invariant loop 1 of the human U5 spliceosomal RNA. The U85 is the first example of a snoRNA that directs modification of an RNA polymerase II-transcribed spliceosomal RNA and that functions both in RNA pseudouridylation and 2'-O-methylation.