snoRNA Gene Cluster Research Articles

Loss of the imprinted, non-coding Snord116 gene cluster in the interval deleted in the Prader Willi syndrome results in murine neuronal and endocrine pancreatic developmental phenotypes.

Global neurodevelopmental delay is a prominent characteristic of individuals with Prader-Willi syndrome (PWS). The neuromolecular bases for these delays are unknown. We identified neuroanatomical changes in the brains of mice deficient for a gene in the minimal critical deletion region for PWS (Snord116p-/m+). In Snord116p-/m+ mice, reduced primary forebrain neuron cell body size is apparent in embryonic day 15.5 fetuses, and persists until postnatal day 30 in cerebellar Purkinje neurons. Snord116 is a snoRNA gene cluster of unknown function that can localize to the nucleolus. In cerebellar Purkinje neurons from postnatal day 30 Snord116p-/m+ mice the reduction in neuronal cell body size was associated with decreased neuronal nucleolar size. We also identified developmental changes in the endocrine pancreas of Snord116p-/m+ animals that persist into adulthood. Mice lacking Snord116 have smaller pancreatic islets; within the islet the percentage of δ-cells is increased, while the percentage of α-cells is reduced. The α-cell markers, Sst and Hhex, are upregulated in Snord116p-/m+ isolated islets while Ins1, Ins2, Pdx1, Nkx6-1, and Pax6 are downregulated. There is a 3-fold increase in the percentage of polyhormonal cells in the neonatal pancreata of Snord116p-/m+ mice, due primarily to an increase in cells co-positive with somatostatin. Snord116 may play a role in islet cell lineage specification. The Snord116 gene cluster is important for developmental processes in the brain as well as the endocrine pancreas.

Small mosaic deletion encompassing the snoRNAs and SNURF‐SNRPN results in an atypical Prader–Willi syndrome phenotype

Genetic analyses were performed in a male patient with suspected Prader-Willi syndrome who presented with hypogonadism, excessive eating, central obesity, small hands and feet and cognition within the low normal range. However, he had no neonatal hypotonia or feeding problems during infancy. Chromosome analysis showed a normal male karyotype. Further analysis with array-CGH identified a mosaic 847 kb deletion in 15q11-q13, including SNURF-SNRPN, the snoRNA gene clusters SNORD116 (HBII-85), SNORD115, (HBII-52), SNORD109 A and B (HBII-438A and B), SNORD64 (HBII-13), and NPAP1 (C15ORF2). MLPA confirmed the deletion and the results were compatible with a paternal origin. Metaphase-FISH verified the mosaicism with the deletion present in 58% of leukocytes analyzed. Three smaller deletions in this region have previously been reported in patients with Prader-Willi syndrome phenotype. All three deletions included SNORD116, but only two encompassed parts of SNURF-SNRPN, implicating SNORD116 as the major contributor to the Prader-Willi phenotype. Our case adds further information about genotype-phenotype correlation and supports the hypothesis that SNORD116 plays a major role in the pathogenesis of Prader-Willi syndrome. Furthermore, it examplifies diagnostic difficulties in atypical cases and illustrates the need for additional testing methods when Prader-Willi syndrome is suspected.

Splicing-Related Features of Introns Serve to Propel Evolution

The role of spliceosomal intronic structures played in evolution has only begun to be elucidated. Comparative genomic analyses of fungal snoRNA sequences, which are often contained within introns and/or exons, revealed that about one-third of snoRNA-associated introns in three major snoRNA gene clusters manifested polymorphisms, likely resulting from intron loss and gain events during fungi evolution. Genomic deletions can clearly be observed as one mechanism underlying intron and exon loss, as well as generation of complex introns where several introns lie in juxtaposition without intercalating exons. Strikingly, by tracking conserved snoRNAs in introns, we found that some introns had moved from one position to another by excision from donor sites and insertion into target sties elsewhere in the genome without needing transposon structures. This study revealed the origin of many newly gained introns. Moreover, our analyses suggested that intron-containing sequences were more prone to sustainable structural changes than DNA sequences without introns due to intron's ability to jump within the genome via unknown mechanisms. We propose that splicing-related structural features of introns serve as an additional motor to propel evolution.

Clustered organization, polycistronic transcription, and evolution of modification-guide snoRNA genes in Euglena gracilis

Previous studies have shown that the eukaryotic microbe Euglena gracilis contains an unusually large assortment of small nucleolar RNAs (snoRNAs) and ribosomal RNA (rRNA) modification sites. However, little is known about the evolutionary mechanisms contributing to this situation. In this study, we have examined the organization and evolution of snoRNA genes in Euglena with the additional objective of determining how these properties relate to the rRNA modification pattern in this protist. We have identified and extensively characterized a clustered pattern of genes encoding previously biochemically isolated snoRNA sequences in E. gracilis. We show that polycistronic transcription is a prevalent snoRNA gene expression strategy in this organism. Further, we have identified 121 new snoRNA coding regions through sequence analysis of these clusters. We have identified an E. gracilis U14 snoRNA homolog clustered with modification-guide snoRNA genes. The U14 snoRNAs in other eukaryotic organisms examined to date typically contain both a modification and a processing domain. E. gracilis U14 lacks the modification domain but retains the processing domain. Our analysis of U14 structure and evolution in Euglena and other eukaryotes allows us to propose a model for its evolution and suggest its processing role may be its more important function, explaining its conservation in many eukaryotes. The preponderance of apparent small and larger-scale duplication events in the genomic regions we have characterized in Euglena provides a mechanism for the generation of the unusually diverse collection and abundance of snoRNAs and modified rRNA sites. Our findings provide the framework for more extensive whole genome analysis to elucidate whether these snoRNA gene clusters are spread across multiple chromosomes and/or form dense "arrays" at a limited number of chromosomal loci.

Prader Willi syndrome

Note The Prader Willi syndrome (PWS) is characterized by diminished fetal activity, dysmorphic facial features, small hands and feet, marked hypotonia, neonatal feeding problems, thick saliva, hyperphagia and weight gain between the ages of 1 and 6, poor linear growth, short stature, hypothalamic dysfunction (obesity, absence of satiety, hypogonadism with cryptorchidism, abnormal temperature control and GH deficiency), cognitive difficulties and characteristic behavioural traits. Inheritance It is sporadic and familial cases are rare. The incidence is 1:10.000-15.000 births. Etiology PWS is genetically heterogeneous. The absent expression of the paternal activity in the critical region on chromosome 15 has been found in patients with PWS. In 70-75% of patients there is a deletion of the paternal 15q11-q13 chromosome (del15) and in about 25% there is a maternal uniparental disomy 15 (UDP15), and a small percentage of patients may have an imprinting center mutation or translocations involving chromosome15. In the 15q11-q13 region a lot of candidate genes are present. The C/D box small nucleolar RNA (snoRNA) gene cluster HBII-85, IPW, PAR1, MAGEL2 and SNRPN genes is not expressed in patients with PWS and may be involved in the phenotype.

Positive Selection and Gene Conversion Drive the Evolution of a Brain-Expressed snoRNAs Cluster

HBII-52 small nucleolar RNAs (snoRNAs) are brain-expressed posttranscriptional modifiers of serotonin receptor 2C RNA. They are organized in a cluster of 47 highly homologous gene copies spanning 100 kb at chromosome 15q11.2. Nucleotide diversity at HBII-52 snoRNA gene cluster in African and European descent populations was analyzed via resequencing of 25 functional snoRNA gene copies. Ninety-four variants were detected, from which 74 are novel. Only 16 variants are shared between Africans and Europeans. We also report a novel Yoruba-specific copy-number variant representing a 5.2-kb polymorphic deletion and resulting in a chimerical functional snoRNA copy. In both populations, the snoRNA genes are characterized by high density of single nucleotide polymorphisms and an excess of low-frequency variants. However, the variability patterns are strictly population specific and there is an extreme divergence in allele frequencies in both resequencing and HapMap data. Several tests of neutrality strongly suggest that the observed extreme population divergence at the HBII-52 region results from positive selection in Europeans. Our analysis of HBII-52 nucleotide variability spectrum shows that gene conversion is the main factor introducing variability at the cluster. Sixty-five substitutions (69%) correspond to a paralogous sequence variant (PSV) in another copy and occur at potential gene conversion tracts of >5 bp. We detected several interparalogue gene-conversion events that involve more than one PSV, with individual frequency patterns suggestive of recurrent gene conversion. Analysis based on derived and ancestral allele distribution shows that gene conversion is at least twice more frequent than point mutations. Gene conversion is an important factor in disrupting patterns of linkage disequilibrium (LD) at short scales. Consistent with this, we detect punctual breaks of LD at gene conversion sites while the overall LD at the HBII-52 cluster is high in both study populations.

Genomewide Analysis of Box C/D and Box H/ACA snoRNAs in Chlamydomonas reinhardtii Reveals an Extensive Organization Into Intronic Gene Clusters

Chlamydomonas reinhardtii is a unicellular green alga, the lineage of which diverged from that of land plants >1 billion years ago. Using the powerful small nucleolar RNA (snoRNA) mining platform to screen the C. reinhardtii genome, we identified 322 snoRNA genes grouped into 118 families. The 74 box C/D families can potentially guide methylation at 96 sites of ribosomal RNAs (rRNAs) and snRNAs, and the 44 box H/ACA families can potentially guide pseudouridylation at 62 sites. Remarkably, 242 of the snoRNA genes are arranged into 76 clusters, of which 77% consist of homologous genes produced by small local tandem duplications. At least 70 snoRNA gene clusters are found within introns of protein-coding genes. Although not exhaustive, this analysis reveals that C. reinhardtii has the highest number of intronic snoRNA gene clusters among eukaryotes. The prevalence of intronic snoRNA gene clusters in C. reinhardtii is similar to that of rice but in contrast with the one-snoRNA-per-intron organization of vertebrates and fungi and with that of Arabidopsis thaliana in which only a few intronic snoRNA gene clusters were identified. This analysis of C. reinhardtii snoRNA gene organization shows the functional importance of introns in a single-celled organism and provides evolutionary insight into the origin of intron-encoded RNAs in the plant lineage.

Deletion of the MBII-85 snoRNA Gene Cluster in Mice Results in Postnatal Growth Retardation

Prader-Willi syndrome (PWS [MIM 176270]) is a neurogenetic disorder characterized by decreased fetal activity, muscular hypotonia, failure to thrive, short stature, obesity, mental retardation, and hypogonadotropic hypogonadism. It is caused by the loss of function of one or more imprinted, paternally expressed genes on the proximal long arm of chromosome 15. Several potential PWS mouse models involving the orthologous region on chromosome 7C exist. Based on the analysis of deletions in the mouse and gene expression in PWS patients with chromosomal translocations, a critical region (PWScr) for neonatal lethality, failure to thrive, and growth retardation was narrowed to the locus containing a cluster of neuronally expressed MBII-85 small nucleolar RNA (snoRNA) genes. Here, we report the deletion of PWScr. Mice carrying the maternally inherited allele (PWScrm−/p+) are indistinguishable from wild-type littermates. All those with the paternally inherited allele (PWScrm+/p−) consistently display postnatal growth retardation, with about 15% postnatal lethality in C57BL/6, but not FVB/N crosses. This is the first example in a multicellular organism of genetic deletion of a C/D box snoRNA gene resulting in a pronounced phenotype.

Elucidating the Role of C/D snoRNA in rRNA Processing and Modification inTrypanosoma brucei

Most eukaryotic C/D small nucleolar RNAs (snoRNAs) guide 2'-O methylation (Nm) on rRNA and are also involved in rRNA processing. The four core proteins that bind C/D snoRNA in Trypanosoma brucei are fibrillarin (NOP1), NOP56, NOP58, and SNU13. Silencing of NOP1 by RNA interference identified rRNA-processing and modification defects that caused lethality. Systematic mapping of 2'-O-methyls on rRNA revealed the existence of hypermethylation at certain positions of the rRNA in the bloodstream form of the parasites, suggesting that this modification may assist the parasites in coping with the major temperature changes during cycling between their insect and mammalian hosts. The rRNA-processing defects of NOP1-depleted cells suggest the involvement of C/D snoRNA in trypanosome-specific rRNA-processing events to generate the small rRNA fragments. MRP RNA, which is involved in rRNA processing, was identified in this study in one of the snoRNA gene clusters, suggesting that trypanosomes utilize a combination of unique C/D snoRNAs and conserved snoRNAs for rRNA processing.

Identification of six new box C/D snoRNA gene clusters from rice

Here we have identified 18 different box C/D snoRNAs encoded in six new gene clusters by the screening of Oryza sativa (rice) genome sequences using a computer program that we have designed to search for non-canonical box C and D sequences. Among this snoRNA collection, five are homologues of snoRNAs previously reported in other plants. One snoRNA is implicated to guide methylation at a corresponding site also modified in yeast rRNA but not in any other plants species yet examined. The other 12 snoRNAs are implicated in guiding 12 completely novel rRNA 2'-O-ribose methylation sites. We have verified the methylation of these rRNA sites by dNTP-concentration dependent primer extension assay. The expression of all snoRNAs reported here was experimentally confirmed by primer extension assay. Polycistronic precursor transcripts from the gene clusters have been demonstrated by RT-PCR. Thus it is highly likely that each of the rice snoRNA gene clusters identified here is transcribed in a polycistronic way, regardless of whether or not they are encoded within introns. This indicates that the mode of polycistronic expression of snoRNA clusters is widespread in rice, as is the case in Arabidopsis thaliana.

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.

Lack of Pwcr1/MBII-85 snoRNA is critical for neonatal lethality in Prader–Willi syndrome mouse models

Prader-Willi syndrome (PWS) is a neurobehavioral disorder caused by the lack of paternal expression of imprinted genes in the human chromosome region 15q11-13. Recent studies of rare human translocation patients narrowed the PWS critical genes to a 121-kb region containing PWCR1/HBII-85 and HBII-438 snoRNA genes. The existing mouse models of PWS that lack the expression of multiple genes, including Snrpn, Ube3a, and many intronic snoRNA genes, are characterized by 80%-100% neonatal lethality. To define the candidate region for PWS-like phenotypes in mice, we analyzed the expression of several genetic elements in mice carrying the large radiation-induced p(30PUb) deletion that includes the p locus. Mice having inherited this deletion from either parent develop normally into adulthood. By Northern blot and RT-PCR assays of brain tissue, we found that Pwcr1/MBII-85 snoRNAs are expressed normally, while MBII-52 snoRNAs are not expressed when the deletion is paternally inherited. Mapping of the distal deletion breakpoint indicated that the p30PUb deletion includes the entire MBII-52 snoRNA gene cluster and three previously unmapped EST sequences. The lack of expression of these elements in mice with a paternal p30PUb deletion indicates that they are not critical for the neonatal lethality observed in PWS mouse models. In addition, we identified MBII-436, the mouse homolog of the HBII-436 snoRNA, confirmed its imprinting status, and mapped it outside of the p30PUb deletion. Taking together all available data, we conclude that the lack of Pwcr1/MBII-85 snoRNA expression is the most likely cause for the neonatal lethality in PWS model mice.

Exclusion of the C/D box snoRNA gene cluster HBII-52 from a major role in Prader?Willi syndrome

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurogenetic disorders caused by the loss of function of imprinted genes in 15q11-q13. The maternally expressed UBE3A gene is affected in AS. Four protein-encoding genes (MKRN3, MAGEL2, NDN and SNURF-SNRPN) and several small nucleolar (sno) RNA genes (HBII-13, HBII-436, HBII-85, HBII-438A, HBII-438B and HBII-52) are expressed from the paternal chromosome only but their contribution to PWS is unclear. To examine the role of the HBII-52 snoRNA genes, we have reinvestigated an AS family with a submicroscopic deletion spanning UBE3A and flanking sequences. By fine mapping of the centromeric deletion breakpoint in this family, we have found that the deletion affects all of the 47 HBII-52 genes. Since the complete loss of the HBII-52 genes in family members who carry the deletion on their paternal chromosome is not associated with an obvious clinical phenotype, we conclude that HBII-52 snoRNA genes do not play a major role in PWS. However, we cannot exclude the possibility that the loss of HBII-52 has a phenotypic effect when accompanied by the loss of function of other genes in 15q11-q13.

A novel gene organization: intronic snoRNA gene clusters from Oryza sativa

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.

Identification and characterization of a novel U14 small nucleolar RNA gene cluster in Oryza sativa

Small nucleolar RNAs (snoRNAs) are required for ribose 2′-O-methylation of eukaryotic ribosomal RNA. Through computer search in international rice genome database, a novel U14 snoRNA gene cluster, consisting of two U14 snoRNA gene candidates, was found on rice chromosome II. They both have box C/D sequences and a 14 nucleotides (nt)-long complementarity to rice 18S ribosomal RNA (rRNA). Functional analysis of this gene cluster indicated that both were transcribed in vivo and might guide the methylations of C418 in rice 18S rRNA. By using primer extension, 5′ and 3′ rapid amplification of cDNA ends, the 5′ and 3′ ends of two snoRNAs were determined. The 52 nt long intergenic spacer of the gene cluster is rich in uridine. The absence of a conserved promoter element in this spacer, the proximity of the genes and the detection of transcripts containing linked U14 snoRNAs by reverse transcript polymerase chain reaction suggest that the rice U14 snoRNAs encoded in the cluster are transcribed as a polycistron under an upstream promoter, and individual U14 snoRNAs are released after processing of the precursor RNAs.

Identification of 10 novel snoRNA gene clusters from Arabidopsis thaliana.

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.

The genes for small nucleolar RNAs in Trypanosoma brucei are organized in clusters and are transcribed as a polycistronic RNA.

Because the organization of snoRNA genes in vertebrates, plants and yeast is diverse, we investigated the organization of snoRNA genes in a distantly related organism, Trypanosoma brucei. We have characterized the second example of a snoRNA gene cluster that is tandemly repeated in the T.BRUCEI: genome. The genes encoding the box C/D snoRNAs TBR12, TBR6, TBR4 and TBR2 make up the cluster. In a genomic organization unique to trypanosomes, there are at least four clusters of these four snoRNA genes tandemly repeated in the T. BRUCEI: genome. We show for the first time that the genes encoding snoRNAs in both this cluster and the SLA cluster are transcribed in an unusual way as a polycistronic RNA.

A novel snoRNA gene cluster in yeast is transcribed as polycistronic pre-snoRNAs

Small nucleolar RNAs (snoRNAs) play an important role in eukaryotic rRNA biogenesis. By combination of a computer search of EMBL database and experimental procedure, a novel snoRNA coding sequence (Z8) was screened out and characterized from yeastSaccharomyces cerevisiue genome. Z8 snoRNA gene codes a boxC/D antisense snoRNA which guides, deduced from structure analysis, the 2'-O-ribose methylation at U(2421) of 25S rRNA. After disruption of Z8 snoRNA gene, the methylation at corresponding site was abolished, but no gmwth delay was observed in various cultural temperatures. Z8 DNA is the first gene of a gene cluster consisting of three cognate snoRNA genes which are located on an intergenic region of chromosome XIII. This gene cluster is co-transcribed as a polycistronic precursor from a + 247 bp U snoRNA gene promoter, followed by processing to release individual snoRNAs, representing a new expression pattern of snoRNA genes.

Seven novel methylation guide small nucleolar RNAs are processed from a common polycistronic transcript by Rat1p and RNase III in yeast.

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.