snoRNA Family Research Articles

EXPRESSION OF H/ACA snoRNA IN CELL LINES WITH CHROMOSOMAL ABNORMALITIES AFTER IRRADIATION

The H/ACA snoRNA family is involved in pseudouridine biogenesis. It prevents genetic changes in cells and makes them more stable due to ribosomal RNA characteristics. Therefore, the study of H/ACA snoRNA expression in cell lines with chromosomal disorders after irradiation is of particular interest.
 The purpose of the study is to analyze the effect of chromosomal disorders on H/ACA snoRNA expression in radioresistant K562 and radiosensitive HL-60 cell lines after radiation exposure.
 Materials and Methods. K562 and HL-60 cell lines were exposed to radiation (4 Gy). H/ACA snoRNA expression was analyzed by NGS sequencing (1, 4, and 24 hours after irradiation).
 Results. The authors revealed differences in H/ACA snoRNA expression by chromosomes in the studied cell lines, as well as the impact of chromosomal abnormalities on H/ACA snoRNA expression after radiation exposure. Changes in the copy number of normal chromosomes lead to minor changes in H/ACA snoRNA expression. Marker chromosomes disrupt H/ACA snoRNA expression. Thus, is becomes impossible to use H/ACA snoRNAs located in abnormal chromosomes as radioresistance markers. Moreover, marker chromosomes decrease the number of H/ACA snoRNAs expressed in K562, despite the greater amount of genetic material.

Small but Mighty-The Emerging Role of snoRNAs in Hematological Malignancies.

Over recent years, the long known class of small nucleolar RNAs (snoRNAs) have gained interest among the scientific community, especially in the clinical context. The main molecular role of this interesting family of non-coding RNAs is to serve as scaffolding RNAs to mediate site-specific RNA modification of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). With the development of new sequencing techniques and sophisticated analysis pipelines, new members of the snoRNA family were identified and global expression patterns in disease backgrounds could be determined. We will herein shed light on the current research progress in snoRNA biology and their clinical role by influencing disease outcome in hematological diseases. Astonishingly, in recent studies snoRNAs emerged as potent biomarkers in a variety of these clinical setups, which is also highlighted by the frequent deregulation of snoRNA levels in the hema-oncological context. However, research is only starting to reveal how snoRNAs might influence cellular functions and the connected disease hallmarks in hematological malignancies.

SnoRNA copy regulation affects family size, genomic location and family abundance levels

BackgroundSmall nucleolar RNAs (snoRNAs) are an abundant class of noncoding RNAs present in all eukaryotes and best known for their involvement in ribosome biogenesis. In mammalian genomes, many snoRNAs exist in multiple copies, resulting from recombination and retrotransposition from an ancestral snoRNA. To gain insight into snoRNA copy regulation, we used Rfam classification and normal human tissue expression datasets generated using low structure bias RNA-seq to characterize snoRNA families.ResultsWe found that although box H/ACA families are on average larger than box C/D families, the number of expressed members is similar for both types. Family members can cover a wide range of average abundance values, but importantly, expression variability of individual members of a family is preferred over the total variability of the family, especially for box H/ACA snoRNAs, suggesting that while members are likely differentially regulated, mechanisms exist to ensure uniformity of the total family abundance across tissues. Box C/D snoRNA family members are mostly embedded in the same host gene while box H/ACA family members tend to be encoded in more than one different host, supporting a model in which box C/D snoRNA duplication occurred mostly by cis recombination while box H/ACA snoRNA families have gained copy members through retrotransposition. And unexpectedly, snoRNAs encoded in the same host gene can be regulated independently, as some snoRNAs within the same family vary in abundance in a divergent way between tissues.ConclusionsSnoRNA copy regulation affects family sizes, genomic location of the members and controls simultaneously member and total family abundance to respond to the needs of individual tissues.

Single Nucleus RNA-Sequencing Reveals Overall Conservation of Hypothalamic Cell Identities but Differential Expression of Specific Genes in the Magel2 Null Mouse Model of Prader-Willi Syndrome

Prader-Willi syndrome (PWS) is a genetic disorder affecting 1 in 10,000 to 30,000 live births. Diagnostic features of PWS including insatiable appetite and obesity are well-defined and many are associated with disruption of hypothalamic function. PWS is caused by sporadic or inherited loss of expression from the paternal allele of one or more maternally imprinted/silenced genes located in chromosomal region 15q11-q13 that encompasses five protein coding genes Mkrn3, Magel2, Necdin and Snurf-Snprn and a family of snoRNAs. Seminal studies have indicated that isolated Magel2 gene silencing plays a pivotal role in the development of many, but not all, clinical features of PWS. Magel2 is highly expressed in the hypothalamus and loss of function studies revealed substantial cellular and molecular changes in hypothalamic neurons located in the suprachiasmatic, paraventricular, supraoptic and arcuate nuclei and the lateral hypothalamus. In addition to neuronal alterations, loss of MAGEL2 increases the density and activation of microglia in adult hypothalamus. In the current study, we characterized global changes in hypothalamic gene expression and searched for novel cell populations associated with loss of Magel2 expression using single nucleus RNA sequencing. Single cell nuclei were isolated in two technical replicates per group from hypothalami of adult male and female Magel2-null (C57BL/6-Magel2tm1Stw/J) and wild type sibling mice for the 10X genomics scRNA-seq pipeline. A total of 63,470 cells divided approximately equally by sex and Magel2 genotype were analyzed. Unsupervised cell clustering identified 19 distinct clusters in males (10 neuronal and 9 non-neuronal) and 21 clusters in females (11 neuronal and 10 non-neuronal) based on their transcriptional profiles of signature genes. The percentages of total cells and the transcriptional profiles of each defined cluster from all four combinations of genotype and sex were nearly identical, indicating that loss-of-function of Magel2 does not alter overall cell cluster identities in the hypothalamus. However, a quantitative analysis of gene expression profiles from all individual clusters demonstrated upregulation of a set of genes including Fkbp5, Zbtb16, Htra1, 2900097C17Rik and 1700030F04Rik predominantly in oligodendrocytes and astrocytes in both sexes of Magel2 null mice. In contrast, the majority of down-regulated genes were found in neuronal cell clusters in both sexes of Magel2 null mice. Our current study is the first to characterize cellular and genetic changes in the whole hypothalamus due to the lack of MAGEL2. The data provide a valuable resource for elucidating the regulatory mechanisms of MAGEL2 for the pathogenesis of PWS and shed light on the discovery of new candidate targets for the potential treatment of PWS. This study is supported by the Foundation for Prader-Willi Research and NIH grant R01DK068400.

The yeast C/D box snoRNA U14 adopts a “weak” K-turn like conformation recognized by the Snu13 core protein in solution

Non-coding RNAs associate with proteins to form ribonucleoproteins (RNPs), such as ribosome, box C/D snoRNPs, H/ACA snoRNPs, ribonuclease P, telomerase and spliceosome to ensure cell viability. The assembly of these RNA-protein complexes relies on the ability of the RNA to adopt the correct bound conformation. K-turn motifs represent ubiquitous binding platform for proteins found in several cellular environment. This structural motif has an internal three-nucleotide bulge flanked on its 3′ side by a G•A/A•G tandem pairs followed by one or two non-Watson-Crick pairs, and on its 5′ side by a classical RNA helix. This peculiar arrangement induces a strong curvature of the phosphodiester backbone, which makes it conducive to multiple tertiary interactions. SNU13/Snu13p (Human/Yeast) binds specifically the U14 C/D box snoRNA K-turn sequence motif. This event is the prerequisite to promote the assembly of the RNP, which contains NOP58/Nop58 and NOP56/Nop56 core proteins and the 2′-O-methyl-transferase, Fibrillarin/Nop1p. The U14 small nucleolar RNA is a conserved non-coding RNA found in yeast and vertebrates required for the pre-rRNA maturation and ribose methylation. Here, we report the solution structure of the free U14 snoRNA K-turn motif (kt-U14) as determined by Nuclear Magnetic Resonance. We demonstrate that a major fraction of free kt-U14 adopts a pre-folded conformation similar to protein bound K-turn, even in the absence of divalent ions. In contrast to the kt-U4 or tyrS RNA, kt-U14 displays a sharp bent in the phosphodiester backbone. The U•U and G•A tandem base pairs are formed through weak hydrogen bonds. Finally, we show that the structure of kt-U14 is stabilized upon Snu13p binding. The structure of the free U14 RNA is the first reference example for the canonical motifs of the C/D box snoRNA family.

Convolutional neural networks for classification of alignments of non-coding RNA sequences.

MotivationThe convolutional neural network (CNN) has been applied to the classification problem of DNA sequences, with the additional purpose of motif discovery. The training of CNNs with distributed representations of four nucleotides has successfully derived position weight matrices on the learned kernels that corresponded to sequence motifs such as protein-binding sites.ResultsWe propose a novel application of CNNs to classification of pairwise alignments of sequences for accurate clustering of sequences and show the benefits of the CNN method of inputting pairwise alignments for clustering of non-coding RNA (ncRNA) sequences and for motif discovery. Classification of a pairwise alignment of two sequences into positive and negative classes corresponds to the clustering of the input sequences. After we combined the distributed representation of RNA nucleotides with the secondary-structure information specific to ncRNAs and furthermore with mapping profiles of next-generation sequence reads, the training of CNNs for classification of alignments of RNA sequences yielded accurate clustering in terms of ncRNA families and outperformed the existing clustering methods for ncRNA sequences. Several interesting sequence motifs and secondary-structure motifs known for the snoRNA family and specific to microRNA and tRNA families were identified.Availability and implementationThe source code of our CNN software in the deep-learning framework Chainer is available at http://www.dna.bio.keio.ac.jp/cnn/, and the dataset used for performance evaluation in this work is available at the same URL.

The fungal snoRNAome.

Small nucleolar RNAs (snoRNAs) are essential players in the rRNA biogenesis due to their involvement in the nucleolytic processing of the precursor and the subsequent guidance of nucleoside modifications. Within the kingdom Fungi, merely a few species-specific surveys have explored their snoRNA repertoire. However, the wide range of the snoRNA landscape spanning all major fungal lineages has not been mapped so far, mainly because of missing tools for automatized snoRNA detection and functional analysis. For the first time, we report here a comprehensive inventory of fungal snoRNAs together with a functional analysis and an in-depth investigation of their evolutionary history including innovations, deletions, and target switches. This large-scale analysis, incorporating more than 120 snoRNA families with more than 7700 individual snoRNA sequences, catalogs and clarifies the landscape of fungal snoRNA families, assigns functions to previously orphan snoRNAs, and increases the number of sequences by 450%. We also show that the snoRNAome is subject to ongoing rearrangements and adaptations, e.g., through lineage-specific targets and redundant guiding functions.

Phylogenetic distribution of plant snoRNA families.

BackgroundSmall nucleolar RNAs (snoRNAs) are one of the most ancient families amongst non-protein-coding RNAs. They are ubiquitous in Archaea and Eukarya but absent in bacteria. Their main function is to target chemical modifications of ribosomal RNAs. They fall into two classes, box C/D snoRNAs and box H/ACA snoRNAs, which are clearly distinguished by conserved sequence motifs and the type of chemical modification that they govern. Similarly to microRNAs, snoRNAs appear in distinct families of homologs that affect homologous targets. In animals, snoRNAs and their evolution have been studied in much detail. In plants, however, their evolution has attracted comparably little attention.ResultsIn order to chart the phylogenetic distribution of individual snoRNA families in plants, we applied a sophisticated approach for identifying homologs of known plant snoRNAs across the plant kingdom. In response to the relatively fast evolution of snoRNAs, information on conserved sequence boxes, target sequences, and secondary structure is combined to identify additional snoRNAs. We identified 296 families of snoRNAs in 24 species and traced their evolution throughout the plant kingdom. Many of the plant snoRNA families comprise paralogs. We also found that targets are well-conserved for most snoRNA families.ConclusionsThe sequence conservation of snoRNAs is sufficient to establish homologies between phyla. The degree of this conservation tapers off, however, between land plants and algae. Plant snoRNAs are frequently organized in highly conserved spatial clusters. As a resource for further investigations we provide carefully curated and annotated alignments for each snoRNA family under investigation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3301-2) contains supplementary material, which is available to authorized users.

Changes in snoRNA and snRNA Abundance in the Human, Chimpanzee, Macaque, and Mouse Brain.

Small nuclear and nucleolar RNAs (snRNAs and snoRNAs) are known to be functionally and evolutionarily conserved elements of transcript processing machinery. Here, we investigated the expression evolution of snRNAs and snoRNAs by measuring their abundance in the frontal cortex of humans, chimpanzees, rhesus monkeys, and mice. Although snRNA expression is largely conserved, 44% of the 185 measured snoRNA and 40% of the 134 snoRNA families showed significant expression divergence among species. The snRNA and snoRNA expression divergence included drastic changes unique to humans: A 10-fold elevated expression of U1 snRNA and a 1,000-fold drop in expression of SNORA29. The decreased expression of SNORA29 might be due to two mutations that affect secondary structure stability. Using in situ hybridization, we further localized SNORA29 expression to nucleolar regions of neuronal cells. Our study presents the first observation of snoRNA abundance changes specific to the human lineage and suggests a possible mechanism underlying these changes.

The emerging landscape of small nucleolar RNAs in cell biology.

Small nucleolar RNAs (snoRNAs) are a large class of small noncoding RNAs present in all eukaryotes sequenced thus far. As a family, they have been well characterized as playing a central role in ribosome biogenesis, guiding either the sequence-specific chemical modification of pre-rRNA (ribosomal RNA) or its processing. However, in higher eukaryotes, numerous orphan snoRNAs were described over a decade ago, with no known target or ascribed function, suggesting the possibility of alternative cellular functionality. In recent years, thanks in great part to advances in sequencing methodologies, we have seen many examples of the diversity that exists in the snoRNA family on multiple levels. In this review, we discuss the identification of novel snoRNA members, of unexpected binding partners, as well as the clarification and extension of the snoRNA target space and the characterization of diverse new noncanonical functions, painting a new and extended picture of the snoRNA landscape. Under the deluge of novel features and functions that have recently come to light, snoRNAs emerge as a central, dynamic, and highly versatile group of small regulatory RNAs. WIREs RNA 2015, 6:381–397. doi: 10.1002/wrna.1284

Matching of Soulmates: Coevolution of snoRNAs and Their Targets

Ribosomal and small nuclear RNAs (snRNAs) comprise numerous modified nucleotides. The modification patterns are retained during evolution, making it even possible to project them from yeast onto human. The stringent conservation of modification sites and the slow evolution of rRNAs and snRNAs contradicts the rapid evolution of small nucleolar RNA (snoRNA) sequences. To explain this discrepancy, we investigated the coevolution of snoRNAs and their targeted sites throughout vertebrates. To measure and evaluate the conservation of RNA-RNA interactions, we defined the interaction conservation index (ICI). It combines the quality of individual interaction with the scope of its conservation in a set of species and serves as an efficient measure to evaluate the conservation of the interaction of snoRNA and target. We show that functions of homologous snoRNAs are evolutionarily stable, thus, members of the same snoRNA family guide equivalent modifications. The conservation of snoRNA sequences is high at target binding regions while the remaining sequence varies significantly. In addition to elucidating principles of correlated evolution, we were able, with the help of the ICI measure, to assign functions to previously orphan snoRNAs and to associate snoRNAs as partners to known chemical modifications unassigned to a given snoRNA. Furthermore, we used predictions of snoRNA functions in conjunction with sequence conservation to identify distant homologies. Because of the high overall entropy of snoRNA sequences, such relationships are hard to detect by means of sequence homology search methods alone.

The Trypanosoma brucei telomerase RNA (TER) homologue binds core proteins of the C/D snoRNA family

Trypanosome protozoan parasites are the causative agents of devastating diseases. Trypanosome telomeres grow in an uncontrolled manner and the variant surface glycoprotein (VSG) genes are located in subtelomeric domains. The gene encoding telomerase reverse transcriptase (TERT) was identified and in this study, we describe the Trypanosoma brucei telomerase RNA (TER). TER RNA is bound by the core proteins of the C/D small nucleolar RNA (snoRNA) family and associates with the methyltransferase-associated protein (MTAP), whose homologue binds to mammalian TER. Silencing of TbTER resulted in telomere shortening. This is the first report of a TER that binds the C/D snoRNA core proteins.

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 90days post-coitus (dpc) and then increased slightly during adulthood in Tongcheng pigs, whereas the expression of the same six genes increased slowly from 65dpc 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.

Comparative genomics of eukaryotic small nucleolar RNAs reveals deep evolutionary ancestry amidst ongoing intragenomic mobility

BackgroundSmall nucleolar (sno)RNAs are required for posttranscriptional processing and modification of ribosomal, spliceosomal and messenger RNAs. Their presence in both eukaryotes and archaea indicates that snoRNAs are evolutionarily ancient. The location of some snoRNAs within the introns of ribosomal protein genes has been suggested to belie an RNA world origin, with the exons of the earliest protein-coding genes having evolved around snoRNAs after the advent of templated protein synthesis. Alternatively, this intronic location may reflect more recent selection for coexpression of snoRNAs and ribosomal components, ensuring rRNA modification by snoRNAs during ribosome synthesis. To gain insight into the evolutionary origins of this genetic organization, we examined the antiquity of snoRNA families and the stability of their genomic location across 44 eukaryote genomes.ResultsWe report that dozens of snoRNA families are traceable to the Last Eukaryotic Common Ancestor (LECA), but find only weak similarities between the oldest eukaryotic snoRNAs and archaeal snoRNA-like genes. Moreover, many of these LECA snoRNAs are located within the introns of host genes independently traceable to the LECA. Comparative genomic analyses reveal the intronic location of LECA snoRNAs is not ancestral however, suggesting the pattern we observe is the result of ongoing intragenomic mobility. Analysis of human transcriptome data indicates that the primary requirement for hosting intronic snoRNAs is a broad expression profile. Consistent with ongoing mobility across broadly-expressed genes, we report a case of recent migration of a non-LECA snoRNA from the intron of a ubiquitously expressed non-LECA host gene into the introns of two LECA genes during the evolution of primates.ConclusionsOur analyses show that snoRNAs were a well-established family of RNAs at the time when eukaryotes began to diversify. While many are intronic, this association is not evolutionarily stable across the eukaryote tree; ongoing intragenomic mobility has erased signal of their ancestral gene organization, and neither introns-first nor evolved co-expression adequately explain our results. We therefore present a third model — constrained drift — whereby individual snoRNAs are intragenomically mobile and may occupy any genomic location from which expression satisfies phenotype.

Characterization of RNase MRP RNA and novel snoRNAs from Giardia intestinalis and Trichomonas vaginalis

BackgroundEukaryotic cells possess a complex network of RNA machineries which function in RNA-processing and cellular regulation which includes transcription, translation, silencing, editing and epigenetic control. Studies of model organisms have shown that many ncRNAs of the RNA-infrastructure are highly conserved, but little is known from non-model protists. In this study we have conducted a genome-scale survey of medium-length ncRNAs from the protozoan parasites Giardia intestinalis and Trichomonas vaginalis.ResultsWe have identified the previously 'missing' Giardia RNase MRP RNA, which is a key ribozyme involved in pre-rRNA processing. We have also uncovered 18 new H/ACA box snoRNAs, expanding our knowledge of the H/ACA family of snoRNAs.ConclusionsResults indicate that Giardia intestinalis and Trichomonas vaginalis, like their distant multicellular relatives, contain a rich infrastructure of RNA-based processing. From here we can investigate the evolution of RNA processing networks in eukaryotes.

Plant noncoding RNA gene discovery by “single-genome comparative genomics”

Plant genomes have undergone multiple rounds of duplications that contributed massively to the growth of gene families. The structure of resulting families has been studied in depth for protein-coding genes. However, little is known about the impact of duplications on noncoding RNA (ncRNA) genes. Here we perform a systematic analysis of duplicated regions in the rice genome in search of such ncRNA repeats. We observe that, just like their protein counterparts, most ncRNA genes have undergone multiple duplications that left visible sequence conservation footprints. The extent of ncRNA gene duplication in plants is such that these sequence footprints can be exploited for the discovery of novel ncRNA gene families on a large scale. We developed an SVM model that is able to retrieve likely ncRNA candidates among the 100,000+ repeat families in the rice genome, with a reasonably low false-positive discovery rate. Among the nearly 4000 ncRNA families predicted by this means, only 90 correspond to putative snoRNA or miRNA families. About half of the remaining families are classified as structured RNAs. New candidate ncRNAs are particularly enriched in UTR and intronic regions. Interestingly, 89% of the putative ncRNA families do not produce a detectable signal when their sequences are compared to another grass genome such as maize. Our results show that a large fraction of rice ncRNA genes are present in multiple copies and are species-specific or of recent origin. Intragenome comparison is a unique and potent source for the computational annotation of this major class of ncRNA.

SnoRNAs from the filamentous fungus Neurospora crassa: structural, functional and evolutionary insights.

BackgroundSnoRNAs 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.ResultsFifty 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.ConclusionThis 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.

Cl gene cluster encoding several small nucleolar RNAs: a comparison amongst trypanosomatids

Small nucleolar RNAs (snoRNAs) are small non-coding RNAs that modify RNA molecules such as rRNA and snRNA by guiding 2'-O-ribose methylation (C/D box snoRNA family) and pseudouridylation reactions (H/ACA snoRNA family). H/ACA snoRNAs are also involved in trans-splicing in trypanosomatids. The aims of this work were to characterise the Cl gene cluster that encodes several snoRNAs in Trypanosoma rangeli and compare it with clusters from Trypanosoma cruzi, Trypanosoma brucei, Leishmania major, Leishmania infantum, Leishmania braziliensis and Leptomonas collosoma. The T. rangeli Cl gene cluster is an 801 base pair (bp) repeat sequence that encodes three C/D (Cl1, Cl2 and Cl4) and three H/ACA (Cl3, Cl5 and Cl6) snoRNAs. In contrast to T. brucei, the Cl3 and Cl5 homologues have not been annotated in the Leishmania or T. cruzi genome projects (http//:www.genedb.org). Of note, snoRNA transcribed regions have a high degree of sequence identity among all species and share gene synteny. Collectively, these findings suggest that the Cl cluster could constitute an interesting target for therapeutic (gene silencing) or diagnostic intervention strategies (PCR-derived tools).

Genome-wide searching with base-pairing kernel functions for noncoding RNAs: computational and expression analysis of snoRNA families in Caenorhabditis elegans

Despite the accumulating research on noncoding RNAs (ncRNAs), it is likely that we are seeing only the tip of the iceberg regarding our understanding of the functions and the regulatory roles served by ncRNAs in cellular metabolism, pathogenesis and host-pathogen interactions. Therefore, more powerful computational and experimental tools for analyzing ncRNAs need to be developed. To this end, we propose novel kernel functions, called base-pairing profile local alignment (BPLA) kernels, for analyzing functional ncRNA sequences using support vector machines (SVMs). We extend the local alignment kernels for amino acid sequences in order to handle RNA sequences by using STRAL's; scoring function, which takes into account sequence similarities as well as upstream and downstream base-pairing probabilities, thus enabling us to model secondary structures of RNA sequences. As a test of the performance of BPLA kernels, we applied our kernels to the problem of discriminating members of an RNA family from nonmembers using SVMs. The results indicated that the discrimination ability of our kernels is stronger than that of other existing methods. Furthermore, we demonstrated the applicability of our kernels to the problem of genome-wide search of snoRNA families in the Caenorhabditis elegans genome, and confirmed that the expression is valid in 14 out of 48 of our predicted candidates by using qRT-PCR. Finally, highly expressed six candidates were identified as the original target regions by DNA sequencing.

Identification, expression and functional analysis of U3 snoRNA genes from Neurospora crassa

By screening of a cDNA library constructed with small RNAs from Neurospora crassa and genome analysis, three U3 snoRNAs of 262, 270 and 275 nt were identified and named as NcU3A, NcU3B and NcU3C, respectively. Expression of the three U3 snoRNA genes was further analyzed by Northern blot hybridization. Meanwhile, two 5′-truncated U3 transcripts with different sizes were also identified, indicating a complex posttranscriptional processing of the U3 snoRNAs. In addition to the known functional elements complementary to 18S rRNA, a novel antisense sequence to 26S rRNA was found in NcU3A. A primer extension assay showed that the antisense sequence did not guide 2′- O-ribose methylation of 26S rRNA but probably functioned as an rRNA chaperon. In the N. crassa genome, three U3 snoRNA genes are independently transcribed and each of them contains a small intron. The same intron-inserting site in the U3 snoRNA genes among N. crassa and yeasts shows the ancient origin of the intron in these organisms. Our results provide new insights into the structural and functional diversity of U3 snoRNA family.