Experimental RNomics: identification of 140 candidates for small non-messenger RNAs in the plant Arabidopsis thaliana

BACKGROUND: Genomes from all organisms known to date express two types of RNA molecules: messenger RNAs (mRNAs), which are translated into proteins, and non-messenger RNAs, which function at the RNA level and do not serve as templates for translation. RESULTS: We have generated a specialized cDNA library from Arabidopsis thaliana to investigate the population of small non-messenger RNAs (snmRNAs) sized 50-500 nt in a plant. From this library, we identified 140 candidates for novel snmRNAs and investigated their expression, abundance, and developmental regulation. Based on conserved sequence and structure motifs, 104 snmRNA species can be assigned to novel members of known classes of RNAs (designated Class I snmRNAs), namely, small nucleolar RNAs (snoRNAs), 7SL RNA, U snRNAs, as well as a tRNA-like RNA. For the first time, 39 novel members of H/ACA box snoRNAs could be identified in a plant species. Of the remaining 36 snmRNA candidates (designated Class II snmRNAs), no sequence or structure motifs were present that would enable an assignment to a known class of RNAs. These RNAs were classified based on their location on the A. thaliana genome. From these, 29 snmRNA species located to intergenic regions, 3 located to intronic sequences of protein coding genes, and 4 snmRNA candidates were derived from annotated open reading frames. Surprisingly, 15 of the Class II snmRNA candidates were shown to be tissue-specifically expressed, while 12 are encoded by the mitochondrial or chloroplast genome. CONCLUSIONS: Our study has identified 140 novel candidates for small non-messenger RNA species in the plant A. thaliana and thereby sets the stage for their functional analysis.     

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:     

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.     

Complete genomic structure of human rpS3: identification of functional U15b snoRNA in the fifth intron

Analysis of the complete genomic structure of the human ribosomal protein S3 (rpS3) gene revealed the presence of a functional U15b snoRNA gene in its intron. Human ribosomal protein S3 (rpS3) gene of 6115 bp long has been identified to contain six introns and seven exons in this study. The first and fifth introns of human S3 gene contain functional U15 snoRNA genes. Although Xenopus and Fugu counterparts also have six introns and seven exons, S3 gene of Fugu contains two functional U15 snoRNAs in the fourth and sixth introns and two pseudo genes for U15 snoRNAs in the first and fifth introns. In Xenopus S1 gene encoding ribosomal protein S3, however, three of its six introns contain U15 snoRNA gene sequence. Sequence comparison of the U15 genes from Xenopus, Fugu and human revealed that the regions involved in binding to 28S rRNA and the consensus sequence (C, D and D' boxes) for snoRNAs are highly conserved among those genes from these three species. Human U15a and U15b RNAs which are derived from the first and the fifth introns, respectively, have been identified to be functional by microinjection of human U15a and U15b snoRNAs into Xenopus oocyte. Northern blot and primer extension analyses confirm that human U15b snoRNA is expressed in vivo.     

Systematic identification and characterization of porcine snoRNAs: structural,functional and developmental insights

Small nucleolar RNAs (snoRNAs) are 50‐ to 300‐nt non‐coding RNAs that are involved in critical cellular events, including rRNA/snRNA modification and splicing, ribosome genesis, telomerase formulation and cell proliferation. The identification of snoRNAs in the pig, which is a widely consumed commercial organism that also has important functions in medicine and biology, will enrich the snoRNA kingdom and provide evolutionary clues about snoRNAs. In this study, we performed a systematic identification of snoRNAs in Sus scrofa and obtained 120 candidate snoRNAs, 65 of which were predicted via sequencing from our constructed cDNA library. The others were obtained by computational screening. The primary structural features examined included the sequence length, GC content, conservation of common box motifs and nucleotide diversity. The results indicate that the primary features of H/ACA box snoRNAs are opposite to those of C/D box snoRNAs. Subsequently, based on chromosomal location and host gene determination, we assigned 91 snoRNAs to nine genome organization modes. Gene duplications and translocations are considered to contribute to the high abundant organization in evolution. Functional information about our novel snoRNAs, such as putative targets, modification sites and guide sequences, was predicted by orthologue alignment. A comparative analysis of predicted targets and possible modified loci on U6 snRNA and 5.8S and 18S rRNAs among five species revealed that targets of snoRNA are conserved among species. Furthermore, we performed a quantitative analysis of six representative snoRNA genes in two pig breeds during different developmental stages. Interestingly, all six snoRNAs from one breed expressed in a similar pattern over the tested time points; however, these same six genes had different expression patterns in the other pig breed. Specifically, expression of all six snoRNAs declined significantly from 65 to 90 days post‐coitus (dpc) and then increased slightly during adulthood in Tongcheng pigs, whereas the expression of the same six genes increased slowly from 65 dpc until adulthood in Landrace pigs. This expression pattern suggests that most housekeeping, non‐coding RNAs from a single pig breed may be similarly expressed during development. Our study adds to the knowledge about the snoRNA family by providing the first genome‐wide study of porcine snoRNAs. The comparative analysis of snoRNAs from different pig breeds gave us evolutionary insight into the function of snoRNAs.     

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.     

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.     

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.     

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.     

Fibrillarin genes encode both a conserved nucleolar protein and a novel small nucleolar RNA involved in ribosomal RNA methylation in Arabidopsis thaliana

Fibrillarin is a key nucleolar protein in eukaryotes which associates with box C/D small nucleolar RNAs (snoRNAs) directing 2'-O-ribose methylation of the rRNA. In this study we describe two genes in Arabidopsis thaliana, AtFib1 and AtFib2, encoding nearly identical proteins conserved with eukaryotic fibrillarins. We demonstrate that AtFib1 and AtFib2 proteins are functional homologs of the yeast Nop1p (fibrillarin) and can rescue a yeast NOP1-null mutant strain. Surprisingly, for the first time in plants, we identified two isoforms of a novel box C/D snoRNA, U60.1f and U60.2f, nested in the fifth intron of AtFib1 and AtFib2. Interestingly after gene duplication the host intronic sequences completely diverged, but the snoRNA was conserved, even in other crucifer fibrillarin genes. We show that the U60f snoRNAs accumulate in seedlings and that their targeted residue on the 25 S rRNA is methylated. Our data reveal that the three modes of expression of snoRNAs, single, polycistronic, and intronic, exist in plants and suggest that the mechanisms directing rRNA methylation, dependent on fibrillarin and box C/D snoRNAs, are evolutionarily conserved in plants.     

Intronic U14 snoRNAs of Xenopus laevis are located in two different parent genes and can be processed from their introns during early oogenesis.

U14 is a member of the rapidly growing family of intronic small nucleolar RNAs (snoRNAs) that are involved in pre-rRNA processing and ribosome biogenesis. These snoRNA species are encoded within introns of eukaryotic protein coding genes and are synthesized via an intron processing pathway. Characterization of Xenopus laevis U14 snoRNA genes has revealed that in addition to the anticipated location of U14 within introns of the amphibian hsc70 gene (introns 4, 5 and 7), additional intronic U14 snoRNAs are also found in the ribosomal protein S13 gene (introns 3 and 4). U14 is thus far a unique intronic snoRNA in that it is encoded within two different parent genes of a single organism. Northern blot analysis revealed that U14 snoRNAs accumulate during early oocyte development and are rapidly expressed after the mid-blastula transition of developing embryos. Microinjection of hsc70 pre-mRNAs into developing oocytes demonstrated that oocytes as early as stages II and III are capable of processing U14 snoRNA from the pre-mRNA precursor. The ability of immature oocytes to process intronic snoRNAs is consistent with the observed accumulation of U14 during oocyte maturation and the developmentally regulated synthesis of rRNA during oogenesis.