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1.  Expression and Processing of a Small Nucleolar RNA from the Epstein-Barr Virus Genome 
PLoS Pathogens  2009;5(8):e1000547.
Small nucleolar RNAs (snoRNAs) are localized within the nucleolus, a sub-nuclear compartment, in which they guide ribosomal or spliceosomal RNA modifications, respectively. Up until now, snoRNAs have only been identified in eukaryal and archaeal genomes, but are notably absent in bacteria. By screening B lymphocytes for expression of non-coding RNAs (ncRNAs) induced by the Epstein-Barr virus (EBV), we here report, for the first time, the identification of a snoRNA gene within a viral genome, designated as v-snoRNA1. This genetic element displays all hallmark sequence motifs of a canonical C/D box snoRNA, namely C/C′- as well as D/D′-boxes. The nucleolar localization of v-snoRNA1 was verified by in situ hybridisation of EBV-infected cells. We also confirmed binding of the three canonical snoRNA proteins, fibrillarin, Nop56 and Nop58, to v-snoRNA1. The C-box motif of v-snoRNA1 was shown to be crucial for the stability of the viral snoRNA; its selective deletion in the viral genome led to a complete down-regulation of v-snoRNA1 expression levels within EBV-infected B cells. We further provide evidence that v-snoRNA1 might serve as a miRNA-like precursor, which is processed into 24 nt sized RNA species, designated as v-snoRNA124pp. A potential target site of v-snoRNA124pp was identified within the 3′-UTR of BALF5 mRNA which encodes the viral DNA polymerase. V-snoRNA1 was found to be expressed in all investigated EBV-positive cell lines, including lymphoblastoid cell lines (LCL). Interestingly, induction of the lytic cycle markedly up-regulated expression levels of v-snoRNA1 up to 30-fold. By a computational approach, we identified a v-snoRNA1 homolog in the rhesus lymphocryptovirus genome. This evolutionary conservation suggests an important role of v-snoRNA1 during γ-herpesvirus infection.
Author Summary
Epstein-Barr virus (EBV) infects about 90% of people worldwide and is associated with different types of cancer. So far, only two large virus-encoded non-coding RNAs (EBER1 and EBER2) and 25 microRNAs (miRNAs) have been identified in the EBV genome. In this study, we report identification of the first member of another abundant non-coding RNA class, a small nucleolar RNA (snoRNA), designated as v-snoRNA1. We show that v-snoRNA1 is located in the nucleolus and interacts with the same proteins as reported for canonical eukaryal snoRNAs. Its biological function is consistent with its high conservation in a distantly related simian herpesvirus genome. Interestingly, v-snoRNA1 might serve as a miRNA-like precursor, which is processed into a 24 nt sized RNA species, designated as v-snoRNA124pp. The viral DNA polymerase BALF5 was identified as a potential target for v-snoRNA124pp. Taken together, these experiments strengthen the crucial function of v-snoRNA1 in EBV infection.
doi:10.1371/journal.ppat.1000547
PMCID: PMC2718842  PMID: 19680535
2.  Human miRNA Precursors with Box H/ACA snoRNA Features 
PLoS Computational Biology  2009;5(9):e1000507.
MicroRNAs (miRNAs) and small nucleolar RNAs (snoRNAs) are two classes of small non-coding regulatory RNAs, which have been much investigated in recent years. While their respective functions in the cell are distinct, they share interesting genomic similarities, and recent sequencing projects have identified processed forms of snoRNAs that resemble miRNAs. Here, we investigate a possible evolutionary relationship between miRNAs and box H/ACA snoRNAs. A comparison of the genomic locations of reported miRNAs and snoRNAs reveals an overlap of specific members of these classes. To test the hypothesis that some miRNAs might have evolved from snoRNA encoding genomic regions, reported miRNA-encoding regions were scanned for the presence of box H/ACA snoRNA features. Twenty miRNA precursors show significant similarity to H/ACA snoRNAs as predicted by snoGPS. These include molecules predicted to target known ribosomal RNA pseudouridylation sites in vivo for which no guide snoRNA has yet been reported. The predicted folded structures of these twenty H/ACA snoRNA-like miRNA precursors reveal molecules which resemble the structures of known box H/ACA snoRNAs. The genomic regions surrounding these predicted snoRNA-like miRNAs are often similar to regions around snoRNA retroposons, including the presence of transposable elements, target site duplications and poly (A) tails. We further show that the precursors of five H/ACA snoRNA-like miRNAs (miR-151, miR-605, mir-664, miR-215 and miR-140) bind to dyskerin, a specific protein component of functional box H/ACA small nucleolar ribonucleoprotein complexes suggesting that these molecules have retained some H/ACA snoRNA functionality. The detection of small RNA molecules that share features of miRNAs and snoRNAs suggest that these classes of RNA may have an evolutionary relationship.
Author Summary
The major functions known for RNA were long believed to be either messenger RNAs, which function as intermediates between genes and proteins, or ribosomal RNAs and transfer RNAs which carry out the translation process. In recent years, however, newly discovered classes of small RNAs have been shown to play important cellular roles. These include microRNAs (miRNAs), which can regulate the production of specific proteins, and small nucleolar RNAs (snoRNAs), which recognise and chemically modify specific sequences in ribosomal RNA. Although miRNAs and snoRNAs are currently believed to be generated by different cellular pathways and to function in different cellular compartments, members of these two types of small RNAs display numerous genomic similarities, and a small number of snoRNAs have been shown to encode miRNAs in several organisms. Here we systematically investigate a possible evolutionary relationship between snoRNAs and miRNAs. Using computational analysis, we identify twenty genomic regions encoding miRNAs with highly significant similarity to snoRNAs, both on the level of their surrounding genomic context as well as their predicted folded structure. A subset of these miRNAs display functional snoRNA characteristics, strengthening the possibility that these miRNA molecules might have evolved from snoRNAs.
doi:10.1371/journal.pcbi.1000507
PMCID: PMC2730528  PMID: 19763159
3.  Analysis of small nucleolar RNAs reveals unique genetic features in malaria parasites 
BMC Genomics  2009;10:68.
Background
Ribosome biogenesis is an energy consuming and stringently controlled process that involves hundreds of trans-acting factors. Small nucleolar RNAs (snoRNAs), important components of ribosome biogenesis are non-coding guide RNAs involved in rRNA processing, nucleotide modifications like 2'-O-ribose methylation, pseudouridylation and possibly gene regulation. snoRNAs are ubiquitous and are diverse in their genomic organization, mechanism of transcription and process of maturation. In vertebrates, most snoRNAs are present in introns of protein coding genes and are processed by exonucleolytic cleavage, while in plants they are transcribed as polycistronic transcripts.
Results
This is a comprehensive analysis of malaria parasite snoRNA genes and proteins that have a role in ribosomal biogenesis. Computational and experimental approaches have been used to identify several box C/D snoRNAs from different species of Plasmodium and confirm their expression. Our analyses reveal that the gene for endoribonuclease Rnt1 is absent from Plasmodium falciparum genome, which indicates the existence of alternative pre-rRNA processing pathways. The structural features of box C/D snoRNAs are highly conserved in Plasmodium genus; however, unlike other organisms most parasite snoRNAs are present in single copy. The genomic localization of parasite snoRNAs shows mixed patterns of those observed in plants, yeast and vertebrates. We have localized parasite snoRNAs in untranslated regions (UTR) of mRNAs, and this is an unprecedented and novel genetic feature. Akin to mammalian snoRNAs, those in Plasmodium may also behave as mobile genetic elements.
Conclusion
This study provides a comprehensive overview on trans-acting genes involved in ribosome biogenesis and also a genetic insight into malaria parasite snoRNA genes.
doi:10.1186/1471-2164-10-68
PMCID: PMC2656528  PMID: 19200392
4.  Role of the Box C/D Motif in Localization of Small Nucleolar RNAs to Coiled Bodies and Nucleoli 
Molecular Biology of the Cell  1999;10(7):2131-2147.
Small nucleolar RNAs (snoRNAs) are a large family of eukaryotic RNAs that function within the nucleolus in the biogenesis of ribosomes. One major class of snoRNAs is the box C/D snoRNAs named for their conserved box C and box D sequence elements. We have investigated the involvement of cis-acting sequences and intranuclear structures in the localization of box C/D snoRNAs to the nucleolus by assaying the intranuclear distribution of fluorescently labeled U3, U8, and U14 snoRNAs injected into Xenopus oocyte nuclei. Analysis of an extensive panel of U3 RNA variants showed that the box C/D motif, comprised of box C′, box D, and the 3′ terminal stem of U3, is necessary and sufficient for the nucleolar localization of U3 snoRNA. Disruption of the elements of the box C/D motif of U8 and U14 snoRNAs also prevented nucleolar localization, indicating that all box C/D snoRNAs use a common nucleolar-targeting mechanism. Finally, we found that wild-type box C/D snoRNAs transiently associate with coiled bodies before they localize to nucleoli and that variant RNAs that lack an intact box C/D motif are detained within coiled bodies. These results suggest that coiled bodies play a role in the biogenesis and/or intranuclear transport of box C/D snoRNAs.
PMCID: PMC25425  PMID: 10397754
5.  A novel gene organization: intronic snoRNA gene clusters from Oryza sativa 
Nucleic Acids Research  2002;30(14):3262-3272.
Based on the analysis of structural features and conserved elements, 27 novel snoRNA genes have been identified from rice. All of them belong to the C/D box-containing snoRNA family except for one that belongs to the H/ACA box type. The newly found genes fall into six clusters that comprise at least three snoRNA genes, and in one case as many as nine genes. Interestingly, four of the six clusters are located within the largest intron of a protein coding gene. The majority of intronic snoRNA gene clusters are simply formed by multiple copies of the same species of snoRNA gene that possess the identical functional elements. This implies a possible mechanism of duplication for the origin of repeating snoRNA coding regions in one intron. However, a few intronic snoRNA gene clusters consisting of different snoRNAs species were also observed. Polycistronic precursors from two independently transcribed clusters were demonstrated by RT–PCR and individual snoRNAs processed from the polycistronic precursors were positively determined by reverse transcription assay. Analyses of the intergenic spacers in the clusters showed that, in addition to a very high AT content, the processing signals in rice snoRNA polycistronic transcripts might be different from those of yeast. Our results demonstrate that, in both plants and mammals, numerous snoRNAs can be produced simultaneously from an mRNA precursor of a host gene despite the different arrangements. The intronic snoRNA gene cluster is a novel gene organization, which is so far unique to plants. The conservation of intronic snoRNA gene clusters in plants was further demonstrated by the study of a similar snoRNA gene organization in the first intron of a Hsp70 gene from wild rice and Zizania caduciflora.
PMCID: PMC135747  PMID: 12136108
6.  SnoRNAs from the filamentous fungus Neurospora crassa: structural, functional and evolutionary insights 
BMC Genomics  2009;10:515.
Background
SnoRNAs represent an excellent model for studying the structural and functional evolution of small non-coding RNAs involved in the post-transcriptional modification machinery for rRNAs and snRNAs in eukaryotic cells. Identification of snoRNAs from Neurospora crassa, an important model organism playing key roles in the development of modern genetics, biochemistry and molecular biology will provide insights into the evolution of snoRNA genes in the fungus kingdom.
Results
Fifty five box C/D snoRNAs were identified and predicted to guide 71 2'-O-methylated sites including four sites on snRNAs and three sites on tRNAs. Additionally, twenty box H/ACA snoRNAs, which potentially guide 17 pseudouridylations on rRNAs, were also identified. Although not exhaustive, the study provides the first comprehensive list of two major families of snoRNAs from the filamentous fungus N. crassa. The independently transcribed strategy dominates in the expression of box H/ACA snoRNA genes, whereas most of the box C/D snoRNA genes are intron-encoded. This shows that different genomic organizations and expression modes have been adopted by the two major classes of snoRNA genes in N. crassa . Remarkably, five gene clusters represent an outstanding organization of box C/D snoRNA genes, which are well conserved among yeasts and multicellular fungi, implying their functional importance for the fungus cells. Interestingly, alternative splicing events were found in the expression of two polycistronic snoRNA gene hosts that resemble the UHG-like genes in mammals. Phylogenetic analysis further revealed that the extensive separation and recombination of two functional elements of snoRNA genes has occurred during fungus evolution.
Conclusion
This is the first genome-wide analysis of the filamentous fungus N. crassa snoRNAs that aids in understanding the differences between unicellular fungi and multicellular fungi. As compared with two yeasts, a more complex pattern of methylation guided by box C/D snoRNAs in multicellular fungus than in unicellular yeasts was revealed, indicating the high diversity of post-transcriptional modification guided by snoRNAs in the fungus kingdom.
doi:10.1186/1471-2164-10-515
PMCID: PMC2780460  PMID: 19895704
7.  Sno/scaRNAbase: a curated database for small nucleolar RNAs and cajal body-specific RNAs 
Nucleic Acids Research  2006;35(Database issue):D183-D187.
Small nucleolar RNAs (snoRNAs) and Cajal body-specific RNAs (scaRNAs) are named for their subcellular localization within nucleoli and Cajal bodies (conserved subnuclear organelles present in the nucleoplasm), respectively. They have been found to play important roles in rRNA, tRNA, snRNAs, and even mRNA modification and processing. All snoRNAs fall in two categories, box C/D snoRNAs and box H/ACA snoRNAs, according to their distinct sequence and secondary structure features. Box C/D snoRNAs and box H/ACA snoRNAs mainly function in guiding 2′-O-ribose methylation and pseudouridilation, respectively. ScaRNAs possess both box C/D snoRNA and box H/ACA snoRNA sequence motif features, but guide snRNA modifications that are transcribed by RNA polymerase II. Here we present a Web-based sno/scaRNA database, called sno/scaRNAbase, to facilitate the sno/scaRNA research in terms of providing a more comprehensive knowledge base. Covering 1979 records derived from 85 organisms for the first time, sno/scaRNAbase is not only dedicated to filling gaps between existing organism-specific sno/scaRNA databases that are focused on different sno/scaRNA aspects, but also provides sno/scaRNA scientists with an opportunity to adopt a unified nomenclature for sno/scaRNAs. Derived from a systematic literature curation and annotation effort, the sno/scaRNAbase provides an easy-to-use gateway to important sno/scaRNA features such as sequence motifs, possible functions, homologues, secondary structures, genomics organization, sno/scaRNA gene's chromosome location, and more. Approximate searches, in addition to accurate and straightforward searches, make the database search more flexible. A BLAST search engine is implemented to enable blast of query sequences against all sno/scaRNAbase sequences. Thus our sno/scaRNAbase serves as a more uniform and friendly platform for sno/scaRNA research. The database is free available at .
doi:10.1093/nar/gkl873
PMCID: PMC1669756  PMID: 17099227
8.  Transcriptional Regulation of Ribosome Components Are Determined by Stress According to Cellular Compartments in Arabidopsis thaliana 
PLoS ONE  2011;6(12):e28070.
Plants have to coordinate eukaryotic ribosomes (cytoribosomes) and prokaryotic ribosomes (plastoribosomes and mitoribosomes) production to balance cellular protein synthesis in response to environmental variations. We identified 429 genes encoding potential ribosomal proteins (RP) in Arabidopsis thaliana. Because cytoribosome proteins are encoded by small nuclear gene families, plastid RP by nuclear and plastid genes and mitochondrial RP by nuclear and mitochondrial genes, several transcriptional pathways were attempted to control ribosome amounts. Examining two independent genomic expression datasets, we found two groups of RP genes showing very different and specific expression patterns in response to environmental stress. The first group represents the nuclear genes coding for plastid RP whereas the second group is composed of a subset of cytoribosome genes coding for RP isoforms. By contrast, the other cytoribosome genes and mitochondrial RP genes show less constraint in their response to stress conditions. The two subsets of cytoribosome genes code for different RP isoforms. During stress, the response of the intensively regulated subset leads to dramatic variation in ribosome diversity. Most of RP genes have same promoter structure with two motifs at conserved positions. The stress-response of the nuclear genes coding plastid RP is related with the absence of an interstitial telomere motif known as telo box in their promoters. We proposed a model for the “ribosome code” that influences the ribosome biogenesis by three main transcriptional pathways. The first pathway controls the basal program of cytoribosome and mitoribosome biogenesis. The second pathway involves a subset of cytoRP genes that are co-regulated under stress condition. The third independent pathway is devoted to the control of plastoribosome biosynthesis by regulating both nuclear and plastid genes.
doi:10.1371/journal.pone.0028070
PMCID: PMC3229498  PMID: 22164228
9.  Computational prediction and validation of C/D, H/ACA and Eh_U3 snoRNAs of Entamoeba histolytica 
BMC Genomics  2012;13:390.
Background
Small nucleolar RNAs are a highly conserved group of small RNAs found in eukaryotic cells. Genes encoding these RNAs are diversely located throughout the genome. They are functionally conserved, performing post transcriptional modification (methylation and pseudouridylation) of rRNA and other nuclear RNAs. They belong to two major categories: the C/D box and H/ACA box containing snoRNAs. U3 snoRNA is an exceptional member of C/D box snoRNAs and is involved in early processing of pre-rRNA. An antisense sequence is present in each snoRNA which guides the modification or processing of target RNA. However, some snoRNAs lack this sequence and often they are called orphan snoRNAs.
Results
We have searched snoRNAs of Entamoeba histolytica from the genome sequence using computational programmes (snoscan and snoSeeker) and we obtained 99 snoRNAs (C/D and H/ACA box snoRNAs) along with 5 copies of Eh_U3 snoRNAs. These are located diversely in the genome, mostly in intergenic regions, while some are found in ORFs of protein coding genes, intron and UTRs. The computationally predicted snoRNAs were validated by RT-PCR and northern blotting. The expected sizes were in agreement with the observed sizes for all C/D box snoRNAs tested, while for some of the H/ACA box there was indication of processing to generate shorter products.
Conclusion
Our results showed the presence of snoRNAs in E. histolytica, an early branching eukaryote, and the structural features of E. histolytica snoRNAs were well conserved when compared with yeast and human snoRNAs. This study will help in understanding the evolution of these conserved RNAs in diverse phylogenetic groups.
doi:10.1186/1471-2164-13-390
PMCID: PMC3542256  PMID: 22892049
U3 snoRNA; Guide/ orphan snoRNAs; Entamoeba histolytica
10.  Seven Novel Methylation Guide Small Nucleolar RNAs Are Processed from a Common Polycistronic Transcript by Rat1p and RNase III in Yeast 
Molecular and Cellular Biology  1999;19(2):1144-1158.
Through a computer search of the genome of the yeast Saccharomyces cerevisiae, the coding sequences of seven different box C/D antisense small nucleolar RNAs (snoRNAs) with the structural hallmarks of guides for rRNA ribose methylation have been detected clustered over a 1.4-kb tract in an inter-open reading frame region of chromosome XIII. The corresponding snoRNAs have been positively identified in yeast cells. Disruption of the nonessential snoRNA gene cluster specifically suppressed the seven cognate rRNA ribose methylations but did not result in any growth delay under the conditions of yeast culture tested. The seven snoRNAs are processed from a common polycistronic transcript synthesized from an independent promoter, similar to some plant snoRNAs but in marked contrast with their vertebrate functional homologues processed from pre-mRNA introns containing a single snoRNA. Processing of the polycistronic precursor requires nucleases also involved in rRNA processing, i.e., Rnt1p and Rat1p. After disruption of the RNT1 gene, the yeast ortholog of bacterial RNase III, production of the seven mature snoRNAs was abolished, while the polycistronic snoRNA precursor accumulated. In cells lacking functional Rat1p, an exonuclease involved in the processing of both pre-rRNA and intron-encoded snoRNAs, several processing intermediates of the polycistronic precursor accumulated. This allowed for the mapping in the precursor of the presumptive Rnt1p endonucleolytic cuts which provide entry sites for subsequent exonucleolytic trimming of the pre-snoRNAs. In line with known properties of double-stranded RNA-specific RNase III, pairs of Rnt1p cuts map next to each other on opposite strands of long double-helical stems in the secondary structure predicted for the polycistronic snoRNA precursor.
PMCID: PMC116044  PMID: 9891049
11.  U3 snoRNA genes are multi-copy and frequently linked to U5 snRNA genes in Euglena gracilis§ 
BMC Genomics  2009;10:528.
Background
U3 snoRNA is a box C/D small nucleolar RNA (snoRNA) involved in the processing events that liberate 18S rRNA from the ribosomal RNA precursor (pre-rRNA). Although U3 snoRNA is present in all eukaryotic organisms, most investigations of it have focused on fungi (particularly yeasts), animals and plants. Relatively little is known about U3 snoRNA and its gene(s) in the phylogenetically broad assemblage of protists (mostly unicellular eukaryotes). In the euglenozoon Euglena gracilis, a distant relative of the kinetoplastid protozoa, Southern analysis had previously revealed at least 13 bands hybridizing with U3 snoRNA, suggesting the existence of multiple copies of U3 snoRNA genes.
Results
Through screening of a λ genomic library and PCR amplification, we recovered 14 U3 snoRNA gene variants, defined by sequence heterogeneities that are mostly located in the U3 3'-stem-loop domain. We identified three different genomic arrangements of Euglena U3 snoRNA genes: i) stand-alone, ii) linked to tRNAArg genes, and iii) linked to a U5 snRNA gene. In arrangement ii), the U3 snoRNA gene is positioned upstream of two identical tRNAArg genes that are convergently transcribed relative to the U3 gene. This scenario is reminiscent of a U3 snoRNA-tRNA gene linkage previously described in trypanosomatids. We document here twelve different U3 snoRNA-U5 snRNA gene arrangements in Euglena; in each case, the U3 gene is linked to a downstream and convergently oriented U5 gene, with the intergenic region differing in length and sequence among the variants.
Conclusion
The multiple U3 snoRNA-U5 snRNA gene linkages, which cluster into distinct families based on sequence similarities within the intergenic spacer, presumably arose by genome, chromosome, and/or locus duplications. We discuss possible reasons for the existence of the unusually large number of U3 snoRNA genes in the Euglena genome. Variability in the signal intensities of the multiple Southern hybridization bands raises the possibility that Euglena contains a naturally aneuploid chromosome complement.
doi:10.1186/1471-2164-10-528
PMCID: PMC2784804  PMID: 19917113
12.  Synchronization of cytoplasmic and transferred mitochondrial ribosomal protein gene expression in land plants is linked to Telo-box motif enrichment 
Background
Chloroplasts and mitochondria evolved from the endosymbionts of once free-living eubacteria, and they transferred most of their genes to the host nuclear genome during evolution. The mechanisms used by plants to coordinate the expression of such transferred genes, as well as other genes in the host nuclear genome, are still poorly understood.
Results
In this paper, we use nuclear-encoded chloroplast (cpRPGs), as well as mitochondrial (mtRPGs) and cytoplasmic (euRPGs) ribosomal protein genes to study the coordination of gene expression between organelles and the host. Results show that the mtRPGs, but not the cpRPGs, exhibit strongly synchronized expression with euRPGs in all investigated land plants and that this phenomenon is linked to the presence of a telo-box DNA motif in the promoter regions of mtRPGs and euRPGs. This motif is also enriched in the promoter regions of genes involved in DNA replication. Sequence analysis further indicates that mtRPGs, in contrast to cpRPGs, acquired telo-box from the host nuclear genome.
Conclusions
Based on our results, we propose a model of plant nuclear genome evolution where coordination of activities in mitochondria and chloroplast and other cellular functions, including cell cycle, might have served as a strong selection pressure for the differential acquisition of telo-box between mtRPGs and cpRPGs. This research also highlights the significance of physiological needs in shaping transcriptional regulatory evolution.
doi:10.1186/1471-2148-11-161
PMCID: PMC3212954  PMID: 21668973
13.  The high diversity of snoRNAs in plants: identification and comparative study of 120 snoRNA genes from Oryza sativa 
Nucleic Acids Research  2003;31(10):2601-2613.
Using a powerful computer-assisted analysis strategy, a large-scale search of small nucleolar RNA (snoRNA) genes in the recently released draft sequence of the rice genome was carried out. This analysis identified 120 different box C/D snoRNA genes with a total of 346 gene variants, which were predicted to guide 135 2′-O-ribose methylation sites in rice rRNAs. Though not exhaustive, this analysis has revealed that rice has the highest number of known box C/D snoRNAs among eukaryotes. Interestingly, although many snoRNA genes are conserved between rice and Arabidopsis, almost half of the identified snoRNA genes are rice specific, which may highlight further the differences in rRNA methylation patterns between monocotyledons and dicotyledons. In addition to 76 singletons, 70 clusters involving 270 snoRNA genes were also found in rice. The large number of the novel snoRNA polycistrons found in the introns of rice protein-coding genes is in contrast to the one-snoRNA-per-intron organization of vertebrates and yeast, and of Arabidopsis in which only a few intronic snoRNA gene clusters were identified. Furthermore, due to a high degree of gene duplication, rice snoRNA genes are clearly redundant and exhibit great sequence variation among isoforms, allowing generation of new snoRNAs for selection. Thus, the large snoRNA gene family in plants can serve as an excellent model for a rapid and functional evolution.
PMCID: PMC156054  PMID: 12736310
14.  Unique genes in plants: specificities and conserved features throughout evolution 
Background
Plant genomes contain a high proportion of duplicated genes as a result of numerous whole, segmental and local duplications. These duplications lead up to the formation of gene families, which are the usual material for many evolutionary studies. However, all characterized genomes include single-copy (unique) genes that have not received much attention. Unlike gene duplication, gene loss is not an unspecific mechanism but is rather influenced by a functional selection. In this context, we have established and used stringent criteria in order to identify suitable sets of unique genes present in plant proteomes. Comparisons of unique genes in the green phylum were used to characterize the gene and protein features exhibited by both conserved and species-specific unique genes.
Results
We identified the unique genes within both A. thaliana and O. sativa genomes and classified them according to the number of homologs in the alternative species: none (U{1:0}), one (U{1:1}) or several (U{1:m}). Regardless of the species, all the genes in these groups present some conserved characteristics, such as small average protein size and abnormal intron number. In order to understand the origin and function of unique genes, we further characterized the U{1:1} gene pairs. The possible involvement of sequence convergence in the creation of U{1:1} pairs was discarded due to the frequent conservation of intron positions. Furthermore, an orthology relationship between the two members of each U{1:1} pair was strongly supported by a high conservation in the protein sizes and transcription levels. Within the promoter of the unique conserved genes, we found a number of TATA and TELO boxes that specifically differed from their mean number in the whole genome. Many unique genes have been conserved as unique through evolution from the green alga Ostreococcus lucimarinus to higher plants. Plant unique genes may also have homologs in bacteria and we showed a link between the targeting towards plastids of proteins encoded by plant nuclear unique genes and their homology with a bacterial protein.
Conclusion
Many of the A. thaliana and O. sativa unique genes are conserved in plants for which the ancestor diverged at least 725 million years ago (MYA). Half of these genes are also present in other eukaryotic and/or prokaryotic species. Thus, our results indicate that (i) a strong negative selection pressure has conserved a number of genes as unique in genomes throughout evolution, (ii) most unique genes are subjected to a low divergence rate, (iii) they have some features observed in housekeeping genes but for most of them there is no functional annotation and (iv) they may have an ancient origin involving a possible gene transfer from ancestral chloroplasts or bacteria to the plant nucleus.
doi:10.1186/1471-2148-8-280
PMCID: PMC2576244  PMID: 18847470
15.  Identification of 10 novel snoRNA gene clusters from Arabidopsis thaliana 
Nucleic Acids Research  2001;29(7):1623-1630.
Ten novel small nucleolar RNA (snoRNA) gene clusters, consisting of two or three snoRNA genes, respectively, were identified from Arabidopsis thaliana. Twelve of the 25 snoRNA genes in these clusters are homologous to those of yeast and mammals according to the conserved antisense sequences that guide 2′-O-ribose methylation of rRNA. The remaining 13 snoRNA genes, including two 5.8S rRNA methylation guides, are new genes identified from A.thaliana. Interestingly, seven methylated nucleotides, predicted by novel snoRNAs Z41a–Z46, are methylated neither in yeast nor in vertebrates. Using primer extension at low dNTP concentration the six methylation sites were determined as expected. These snoRNAs were recognized as specific guides for 2′-O-ribose methylation of plant rRNAs. Z42, however, did not guide the expected methylation of 25S rRNA in our assay. Thus, its function remains to be elucidated. The intergenic spacers of the gene clusters are rich in uridine (up to 40%) and most of them range in size from 35 to 100 nt. Lack of a conserved promoter element in each spacer and the determination of polycistronic transcription from a cluster by RT–PCR assay suggest that the snoRNAs encoded in the clusters are transcribed as a polycistron under an upstream promoter, and individual snoRNAs are released after processing of the precursor. Numerous snoRNA gene clusters identified from A.thaliana and other organisms suggest that the snoRNA gene cluster is an ancient gene organization existing abundantly in plants.
PMCID: PMC31268  PMID: 11266566
16.  Computer identification of snoRNA genes using a Mammalian Orthologous Intron Database 
Nucleic Acids Research  2005;33(14):4578-4583.
Based on comparative genomics, we created a bioinformatic package for computer prediction of small nucleolar RNA (snoRNA) genes in mammalian introns. The core of our approach was the use of the Mammalian Orthologous Intron Database (MOID), which contains all known introns within the human, mouse and rat genomes. Introns from orthologous genes from these three species, that have the same position relative to the reading frame, are grouped in a special orthologous intron table. Our program SNO.pl searches for conserved snoRNA motifs within MOID and reports all cases when characteristic snoRNA-like structures are present in all three orthologous introns of human, mouse and rat sequences. Here we report an example of the SNO.pl usage for searching a particular pattern of conserved C/D-box snoRNA motifs (canonical C- and D-boxes and the 6 nt long terminal stem). In this computer analysis, we detected 57 triplets of snoRNA-like structures in three mammals. Among them were 15 triplets that represented known C/D-box snoRNA genes. Six triplets represented snoRNA genes that had only been partially characterized in the mouse genome. One case represented a novel snoRNA gene, and another three cases, putative snoRNAs. Our programs are publicly available and can be easily adapted and/or modified for searching any conserved motifs within mammalian introns.
doi:10.1093/nar/gki754
PMCID: PMC1184218  PMID: 16093549
17.  Mammalian Small Nucleolar RNAs Are Mobile Genetic Elements 
PLoS Genetics  2006;2(12):e205.
Small nucleolar RNAs (snoRNAs) of the H/ACA box and C/D box categories guide the pseudouridylation and the 2′-O-ribose methylation of ribosomal RNAs by forming short duplexes with their target. Similarly, small Cajal body–specific RNAs (scaRNAs) guide modifications of spliceosomal RNAs. The vast majority of vertebrate sno/scaRNAs are located in introns of genes transcribed by RNA polymerase II and processed by exonucleolytic trimming after splicing. A bioinformatic search for orthologues of human sno/scaRNAs in sequenced mammalian genomes reveals the presence of species- or lineage-specific sno/scaRNA retroposons (sno/scaRTs) characterized by an A-rich tail and an ∼14-bp target site duplication that corresponds to their insertion site, as determined by interspecific genomic alignments. Three classes of snoRTs are defined based on the extent of intron and exon sequences from the snoRNA parental host gene they contain. SnoRTs frequently insert in gene introns in the sense orientation at genomic hot spots shared with other genetic mobile elements. Previously characterized human snoRNAs are encoded in retroposons whose parental copies can be identified by phylogenic analysis, showing that snoRTs can be faithfully processed. These results identify snoRNAs as a new family of mobile genetic elements. The insertion of new snoRNA copies might constitute a safeguard mechanism by which the biological activity of snoRNAs is maintained in spite of the risk of mutations in the parental copy. I furthermore propose that retroposition followed by genetic drift is a mechanism that increased snoRNA diversity during vertebrate evolution to eventually acquire new RNA-modification functions.
Synopsis
Large parts of vertebrate genomes are made of repeated sequences that were first considered to be junk DNA, but are now recognized as important actors in genome evolution. Most are genetic mobile elements that can gain additional genomic copies by a copy-and-paste mechanism involving an RNA intermediate. One class, the L1 elements, encodes two proteins required for its integration at new sites. Others, like primate Alu elements, hijack the L1 machinery for their mobilization, and are thus referred to as nonautonomous. In this article, Weber describes a new class of vertebrate nonautonomous mobile elements derived from small nucleolar RNAs (snoRNAs). These nonprotein-coding RNAs are encoded in gene introns and are involved in chemical modifications of selected bases of ribosomal RNAs. The article shows that new snoRNA copies were generated in vertebrate genomes via the copy-and-paste mechanism. Many of them are species-specific, and their insertion point was precisely determined by alignment with the corresponding genomic portion from a neighbour species. The mobilization of snoRNA gene sequences might ensure the presence of a functional copy when the parental one becomes invalidated by mutations. Moreover, such copies could evolve on their own to acquire the capacity of guiding new modifications of ribosomal RNAs.
doi:10.1371/journal.pgen.0020205
PMCID: PMC1687206  PMID: 17154719
18.  Box C/D snoRNP catalysed methylation is aided by additional pre-rRNA base-pairing 
The EMBO Journal  2011;30(12):2420-2430.
Box C/D snoRNP catalysed methylation is aided by additional pre-rRNA base-pairing
Box C/D small nucleolar RNPs catalyse 2′-O-methylation of eukaryotic ribosomal RNA. A large-scale analysis of yeast box C/D snoRNAs reveals conserved ‘extra base-pairing' between snoRNAs and regions adjacent to their rRNA methylation site and points to a role for the non-catalytic protein subunits Nop56 and Nop58 in rRNA binding.
2′-O-methylation of eukaryotic ribosomal RNA (r)RNA, essential for ribosome function, is catalysed by box C/D small nucleolar (sno)RNPs. The RNA components of these complexes (snoRNAs) contain one or two guide sequences, which, through base-pairing, select the rRNA modification site. Adjacent to the guide sequences are protein-binding sites (the C/D or C′/D′ motifs). Analysis of >2000 yeast box C/D snoRNAs identified additional conserved sequences in many snoRNAs that are complementary to regions adjacent to the rRNA methylation site. This ‘extra base-pairing' was also found in many human box C/D snoRNAs and can stimulate methylation by up to five-fold. Sequence analysis, combined with RNA–protein crosslinking in Saccharomyces cerevisiae, identified highly divergent box C′/D′ motifs that are bound by snoRNP proteins. In vivo rRNA methylation assays showed these to be active. Our data suggest roles for non-catalytic subunits (Nop56 and Nop58) in rRNA binding and support an asymmetric model for box C/D snoRNP organization. The study provides novel insights into the extent of the snoRNA–rRNA interactions required for efficient methylation and the structural organization of the snoRNPs.
doi:10.1038/emboj.2011.148
PMCID: PMC3116282  PMID: 21556049
methylation; ribosome; rRNA; snoRNA; snoRNP
19.  Characterisation of the U83 and U84 small nucleolar RNAs: two novel 2′-O-ribose methylation guide RNAs that lack complementarities to ribosomal RNAs 
Nucleic Acids Research  2000;28(6):1348-1354.
In eukaryotic cells, the site-specific 2′-O-ribose methy-lation of ribosomal RNAs (rRNAs) and the U6 spliceosomal small nuclear RNA (snRNA) is directed by small nucleolar RNAs (snoRNAs). The C and D box-containing 2′-O-methylation guide snoRNAs select the correct substrate nucleotide through formation of a long 10–21 bp interaction with the target rRNA and U6 snRNA sequences. Here, we report on the characterisation of two novel mammalian C/D box snoRNAs, called U83 and U84, that contain all the elements that are essential for accumulation and function of 2′-O-methylation guide snoRNAs. However, in contrast to all of the known 2′-O-methylation guide RNAs, the human, mouse and pig U83 and U84 snoRNAs feature no antisense elements complementary to rRNA or U6 snRNA sequences. The human U83 and U84 snoRNAs are not associated with maturing nucleolar pre-ribosomal particles, suggesting that they do not function in rRNA biogenesis. Since artificial substrate RNAs complementary to the evolutionarily conserved putative substrate recognition motifs of the U83 and U84 snoRNAs were correctly 2′-O-methy-lated in the nucleolus of mouse cells, we suggest that the new snoRNAs act as 2′-O-methylation guides for cellular RNAs other then rRNAs and the U6 snRNA.
PMCID: PMC111033  PMID: 10684929
20.  Processing of Intron-Encoded Box C/D Small Nucleolar RNAs Lacking a 5′,3′-Terminal Stem Structure 
Molecular and Cellular Biology  2000;20(13):4522-4531.
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 β-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.
PMCID: PMC85834  PMID: 10848579
21.  Genome-wide analysis of chicken snoRNAs provides unique implications for the evolution of vertebrate snoRNAs 
BMC Genomics  2009;10:86.
Background
Small nucleolar RNAs (snoRNAs) represent one of the largest groups of functionally diverse trans-acting non-protein-coding (npc) RNAs currently known in eukaryotic cells. Chicken snoRNAs have been very poorly characterized when compared to other vertebrate snoRNAs. A genome-wide analysis of chicken snoRNAs is therefore of great importance to further understand the functional evolution of snoRNAs in vertebrates.
Results
Two hundred and one gene variants encoding 93 box C/D and 62 box H/ACA snoRNAs were identified in the chicken genome and are predicted to guide 86 2'-O-ribose methylations and 69 pseudouridylations of rRNAs and spliceosomal RNAs. Forty-four snoRNA clusters were grouped into four categories based on synteny characteristics of the clustered snoRNAs between chicken and human. Comparative analyses of chicken snoRNAs revealed extensive recombination and separation of guiding function, with cooperative evolution between the guiding duplexes and modification sites. The gas5-like snoRNA host gene appears to be a hotspot of snoRNA gene expansion in vertebrates. Our results suggest that the chicken is a good model for the prediction of functional snoRNAs, and that intragenic duplication and divergence might be the major driving forces responsible for expansion of novel snoRNA genes in the chicken genome.
Conclusion
We have provided a detailed catalog of chicken snoRNAs that aids in understanding snoRNA gene repertoire differences between avians and other vertebrates. Our genome-wide analysis of chicken snoRNAs improves annotation of the 'darkness matter' in the npcRNA world and provides a unique perspective into snoRNA evolution in vertebrates.
doi:10.1186/1471-2164-10-86
PMCID: PMC2653536  PMID: 19232134
22.  Human box C/D snoRNAs with miRNA like functions: expanding the range of regulatory RNAs 
Nucleic Acids Research  2010;39(2):675-686.
Small nucleolar RNAs (snoRNAs) and microRNAs are two classes of non-protein-coding RNAs with distinct functions in RNA modification or post-transcriptional gene silencing. In this study, we introduce novel insights to RNA-induced gene activity adjustments in human cells by identifying numerous snoRNA-derived molecules with miRNA-like function, including H/ACA box snoRNAs and C/D box snoRNAs. In particular, we demonstrate that several C/D box snoRNAs give rise to gene regulatory RNAs, named sno-miRNAs here. Our data are complementing the increasing number of studies in the field of small RNAs with regulatory functions. In massively deep sequencing of small RNA fractions we identified high copy numbers of sub-sequences from >30 snoRNAs with lengths of ≥18 nt. RNA secondary structure prediction indicated for a majority of candidates a location in predicted stem regions. Experimental analysis revealed efficient gene silencing for 11 box C/D sno-miRNAs, indicating cytoplasmic processing and recruitment to the RNA silencing machinery. Assays in four different human cell lines indicated variations in both the snoRNA levels and their processing to active sno-miRNAs. In addition we show that box D elements are predominantly flanking at least one of the sno-miRNA strands, while the box C element locates within the sequence of the sno-miRNA guide strand.
doi:10.1093/nar/gkq776
PMCID: PMC3025573  PMID: 20846955
23.  Isolation and Characterization of Three Cassava Elongation Factor 1 Alpha (MeEF1A) Promoters 
PLoS ONE  2014;9(1):e84692.
In plant genetic engineering, the identification of gene promoters leading to particular expression patterns is crucial for the development of new genetically modified plant generations. This research was conducted in order to isolate and characterize several new promoters from cassava (Manihot esculenta Crantz) elongation factor 1 alpha (EF1A) gene family. Three promoters MeEF1A3, MeEF1A4 and MeEF1A5 were successfully isolated. Sequence analyses showed that all of the promoters contain three conserved putative cis-acting elements which are located upstream of the transcription start site. These elements are included a TEF1, a TELO and TATA boxes. In addition, all of the promoters also have the 5′UTR intron but with a different lengths. These promoters were constructed translationally with gusA reporter gene (promoter::gusA fusion) in pBI-121 binary vector to build a new binary vector using Overlap Extension PCR Cloning (OEPC) technique. Transient expression assay that was done by using agroinfiltration method was used to show functionality of these promoters. Qualitative and quantitative analysis from GUS assay showed that these promoters were functional and conferred a specific activity in tobacco seedlings (Nicotiana tabacum), tomato fruits (Solanum lycopersicum) and banana fruits (Musa acuminata). We hypothesized that MeEF1A6 could be categorized as a constitutive promoter because it was able to drive the gene expression in all transformed tissue described in here and also comparable to CaMV35S. On the other hand, MeEF1A3 drove specific expression in the aerial parts of seedlings such as hypocotyl and cotyledon thus MeEF1A5 drove specific expression in fruit tissue. The results obtained from transient analysis showed that these promoters had a distinct activity although they came from same gene family. The DNA sequences identified here are new promoters potentially use for genetic engineering in cassava or other plants.
doi:10.1371/journal.pone.0084692
PMCID: PMC3880305  PMID: 24404183
24.  Accumulation of H/ACA snoRNPs depends on the integrity of the conserved central domain of the RNA-binding protein Nhp2p 
Nucleic Acids Research  2001;29(13):2733-2746.
Box H/ACA small nucleolar ribonucleoprotein particles (H/ACA snoRNPs) play key roles in the synthesis of eukaryotic ribosomes. How box H/ACA snoRNPs are assembled remains unknown. Here we show that yeast Nhp2p, a core component of these particles, directly binds RNA. In vitro, Nhp2p interacts with high affinity with RNAs containing irregular stem–loop structures but shows weak affinity for poly(A), poly(C) or for double-stranded RNAs. The central region of Nhp2p is believed to function as an RNA-binding domain, since it is related to motifs found in various RNA-binding proteins. Removal of two amino acids that shortens a putative β-strand element within Nhp2p central domain impairs the ability of the protein to interact with H/ACA snoRNAs in cell extracts. In vivo, this deletion prevents cell viability and leads to a strong defect in the accumulation of H/ACA snoRNAs and Gar1p. These data suggest that proper direct binding of Nhp2p to H/ACA snoRNAs is required for the assembly of H/ACA snoRNPs and hence for the stability of some of their components. In addition, we show that converting a highly conserved glycine residue (G59) within Nhp2p central domain to glutamate significantly reduces cell growth at 30 and 37°C. Remarkably, this modification affects the steady-state levels of H/ACA snoRNAs and the strength of Nhp2p association with these RNAs to varying degrees, depending on the nature of the H/ACA snoRNA. Finally, we show that the modified Nhp2p protein whose interaction with H/ACA snoRNAs is impaired cannot accumulate in the nucleolus, suggesting that only the assembled H/ACA snoRNP particles can be efficiently retained in the nucleolus.
PMCID: PMC55775  PMID: 11433018
25.  SNOntology: Myriads of novel snornas or just a mirage? 
BMC Genomics  2011;12:543.
Background
Small nucleolar RNAs (snoRNAs) are a large group of non-coding RNAs (ncRNAs) that mainly guide 2'-O-methylation (C/D RNAs) and pseudouridylation (H/ACA RNAs) of ribosomal RNAs. The pattern of rRNA modifications and the set of snoRNAs that guide these modifications are conserved in vertebrates. Nearly all snoRNA genes in vertebrates are localized in introns of other genes and are processed from pre-mRNAs. Thus, the same promoter is used for the transcription of snoRNAs and host genes.
Results
The series of studies by Dahai Zhu and coworkers on snoRNAs and their genes were critically considered. We present evidence that dozens of species-specific snoRNAs that they described in vertebrates are experimental artifacts resulting from the improper use of Northern hybridization. The snoRNA genes with putative intrinsic promoters that were supposed to be transcribed independently proved to contain numerous substitutions and are, most likely, pseudogenes. In some cases, they are localized within introns of overlooked host genes. Finally, an increased number of snoRNA genes in mammalian genomes described by Zhu and coworkers is also an artifact resulting from two mistakes. First, numerous mammalian snoRNA pseudogenes were considered as genes, whereas most of them are localized outside of host genes and contain substitutions that question their functionality. Second, Zhu and coworkers failed to identify many snoRNA genes in non-mammalian species. As an illustration, we present 1352 C/D snoRNA genes that we have identified and annotated in vertebrates.
Conclusions
Our results demonstrate that conclusions based only on databases with automatically annotated ncRNAs can be erroneous. Special investigations aimed to distinguish true RNA genes from their pseudogenes should be done. Zhu and coworkers, as well as most other groups studying vertebrate snoRNAs, give new names to newly described homologs of human snoRNAs, which significantly complicates comparison between different species. It seems necessary to develop a uniform nomenclature for homologs of human snoRNAs in other vertebrates, e.g., human gene names prefixed with several-letter code denoting the vertebrate species.
doi:10.1186/1471-2164-12-543
PMCID: PMC3349704  PMID: 22047601

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