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[Figure: see text].

BACKGROUND Long-non-coding RNA (lncRNA) SNHG15 has been reported to be an aberrantly expressed lncRNA in patients with ischemic stroke, but its role in neuronal injury following ischemic stroke remains unclear. We hypothesized that this lncRNA is associated with the pathogenesis of ischemic stroke. MATERIAL AND METHODS A mouse model of ischemic stroke was established by middle cerebral artery occlusion (MCAO). A neurogenic mouse cell line Neuro-2a (N2a) was subjected to oxygen-glucose deprivation (OGD) for in vitro experiments. Expression of SNHG15, microRNA-18a (miR-18a), and CXCL13 in mouse brain and in OGD-treated N2a cells was determined. Altered expression of SNHG15 and miR-18a was introduced to detect their roles in N2a cell viability and apoptosis. Targeting relationships between miR-18a and SNHG15 or CXCL13 were validated by luciferase assays. Cells were treated with the ERK/MEK antagonist U0126 to assess the role of the ERK/MEK signaling pathway in N2a cell growth. RESULTS SNHG15 and CXCL13 were overexpressed and miR-18a was underexpressed in MCAO-induced mice and OGD-treated N2a cells. Silencing of SNHG15 or overexpression of miR-18a promoted cell viability, while decreased cell apoptosis induced by OGD; however, subsequent disruption of the ERK/MEK signaling pathway reversed these effects. SNHG15 was found to bind to miR-18a, which could further target CXCL13. CONCLUSIONS Silencing of SNHG15 led to CXCL13 upregulation through sequestering miR-18a and the following ERK/MEK activation, thus enhancing viability while reducing apoptosis of N2a cells. SNHG15 may serve as a novel target for ischemic stroke treatment.

Objective: This study aims to investigate whether SNHG16 (small nucleolar RNA host gene 16) can promote the progression of osteoarthritis (OA) by regulating the microRNA-93-5p/Cyclin D1 (CCND1) axis, thereby finding new therapeutic targets for the treatment of OA., Patients and Methods: A total of 23 OA patients and 23 patients undergoing lower extremity amputation were enrolled in this study. We collected their cartilage tissues from knee joint for isolating chondrocytes. The relative levels of SNHG16, CCND1 and microRNA-93-5p in cartilage tissues of OA patients and controls were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The regulatory effect of SNHG16 on proliferative potential of chondrocytes was evaluated by Cell Counting Kit-8 (CCK-8) and colony formation assay, respectively. Cell cycle progression was examined using flow cytometry. Dual-Luciferase reporter gene assay was conducted to verify the binding between SNHG16 with microRNA-93-5p and microRNA-93-5p with CCND1. Rescue experiments were performed to elucidate whether SNHG16 regulated CCND1 expression by targeting microRNA-93-5p., Results: The expressions of SNHG16 and CCND1 upregulated, while microRNA-93-5p downregulated in cartilage tissues of OA patients relative to controls. Correlation regression analyses showed a negative expression correlation between SNHG16 and microRNA-93-5p, as well as CCND1 and microRNA-93-5p in OA patients. On the contrary, SNHG16 expression was positively correlated to CCND1 expression in OA. The knockdown of SNHG16 suppressed viability, cloning ability and cell cycle progression, but induced apoptosis in chondrocytes. Dual-Luciferase reporter gene assay showed that SNHG16 could bind to microRNA-93-5p. SNHG16 knockdown markedly upregulated the expression of microRNA-93-5p. Moreover, the knockdown of microRNA-93-5p reversed the inhibited viability due to SNHG16 knockdown. Transfection of microRNA-93-5p mimics markedly inhibited CCND1 expression. Importantly, CCND1 overexpression reversed the inhibitory effect of SNHG16 knockdown on chondrocyte viability., Conclusions: SNHG16 promotes the development of OA by regulating microRNA-93-5p/CCND1 axis.

Small RNAs (sRNAs) are short noncoding RNAs that play roles in many biological processes, including drought responses in plants. However, how the expression of sRNAs dynamically changes with the gradual imposition of drought stress in plants is largely unknown. We generated time-series sRNA sequence data from maize ( L.) seedlings under drought stress (DS) and under well-watered (WW) conditions at the same time points. Analyses of length, functional annotation, and abundance of 736,372 nonredundant sRNAs from both DS and WW data, as well as genome copy numbers at the corresponding genomic regions, revealed distinct patterns of abundance and genome organization for different sRNA classes. The analysis identified 6646 sRNAs whose regulation was altered in response to drought stress. Among drought-responsive sRNAs, 1325 showed transient downregulation by the seventh day, coinciding with visible symptoms of drought stress. The profiles revealed drought-responsive microRNAs, as well as other sRNAs that originated from ribosomal RNAs (rRNAs), splicing small nuclear RNAs, and small nucleolar RNAs (snoRNA). Expression profiles of their sRNA derivers indicated that snoRNAs might play a regulatory role through regulating the stability of rRNAs and splicing small nuclear RNAs under drought condition., (Copyright © 2019 Crop Science Society of America.)

The pathobiological causes, the shared cellular and molecular pathways in catatonia and in catatonic presentation in neuropsychiatric disorders are yet to be determined. The hypotheses in this paper have been deduced from the latest scientific research findings and clinical observations of patients with genetic disorders, behavioral phenotypes and other family members suffering mental disorders. The first hypothesis postulates that catatonia and the heterogeneity of catatonic signs and symptoms involve nucleolar dysfunction arising from abnormalities of the brain-specific, non-coding micro-RNA, SNORD115 genes (either duplications or deletions) which result in pathobiological dysfunction of various combinations in the downstream pathways (possibly along with other genes in these shared pathways). SNORD115 controls five genes CRHR1, PBRM1, TAF1, DPM2, and RALGPS1 as well as the alternative splicing of serotonin 2C receptor. SNORD115 abnormalities with varying downstream multigene involvement would account for catatonia across the life span within some subtypes of autism spectrum disorders, schizophrenia, bipolar and major depressive disorder, psychosis, genetic disorders, and in immune disorders such as anti-N-methyl-d-aspartate receptor (NMDAR) antibody encephalitis as well as the susceptibility to the neuroleptic malignant syndrome (NMS) if environmentally triggered. Furthermore, SNORD115 genes may underlie a genetic vulnerability when environmental triggers result in excess serotonin producing the serotonin syndrome, a condition similar to NMS in which catatonia may occur. Dysfunction of SNORD115-PBRM1 connecting with SMARCA2 as well as other proven schizophrenia-associated genes might explain why traditionally catatonia has been classified with schizophrenia. SNORD115-TAF1 and SNORD-DPM2 dysfunction introduce possible clues to the parkinsonism and increased creatinine phosphokinase in NMS, while abnormalities of SNORD115-RALGPS1 suggest links to both anti-NMDAR encephalitis and the proven predisposing catatonic SHANK3 gene. The second hypothesis postulates that periodic catatonia (PC) on 15q15 involves abnormalities of vacuolar protein sorting 39 (VPS39), a proven de novo schizophrenic gene in this chromosomal locus and part of the HOPS complex. These will impact the autophagic and endocytic pathways, thereby lowering lysosomal degradation. VPS39 mutations may be considered also to disrupt lysosome-mitochondria tethering and transport of lipids and calcium through membrane contact sites (MCSs). To account for the periodicity in PC it is speculated that the mammalian equivalent of the vacuole and mitochondria patch (vCLAMP) would be altered by VPS39 mutations and subsequently followed by the mammalian equivalent of endoplasmic reticulum mitochondria encounter structure (ERMES) restoring mitochondrial homeostasis. Future precision psychiatry will require accurate pathophysiologically-defined psychiatric diagnoses to accelerate the discovery of specific molecular-targeted medications to improve therapeutic outcomes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

mRNA 3' end processing is an essential step in gene expression. It is well established that canonical eukaryotic pre-mRNA 3' processing is carried out within a macromolecular machinery consisting of dozens of trans-acting proteins. However, it is unknown whether RNAs play any role in this process. Unexpectedly, we found that a subset of small nucleolar RNAs (snoRNAs) are associated with the mammalian mRNA 3' processing complex. These snoRNAs primarily interact with Fip1, a component of cleavage and polyadenylation specificity factor (CPSF). We have functionally characterized one of these snoRNAs and our results demonstrated that the U/A-rich SNORD50A inhibits mRNA 3' processing by blocking the Fip1-poly(A) site (PAS) interaction. Consistently, SNORD50A depletion altered the Fip1-RNA interaction landscape and changed the alternative polyadenylation (APA) profiles and/or transcript levels of a subset of genes. Taken together, our data revealed a novel function for snoRNAs and provided the first evidence that non-coding RNAs may play an important role in regulating mRNA 3' processing., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes-UtpA and UtpB-interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA-protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 5' end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 5' ETS and U3 snoRNA as well as the 3' boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly.

C/D box small nucleolar RNAs (SNORDs) are small noncoding RNAs, and their best-understood function is to target the methyltransferase fibrillarin to rRNA (for example, SNORD27 performs 2'-O-methylation of A27 in 18S rRNA). Unexpectedly, we found a subset of SNORDs, including SNORD27, in soluble nuclear extract made under native conditions, where fibrillarin was not detected, indicating that a fraction of the SNORD27 RNA likely forms a protein complex different from canonical snoRNAs found in the insoluble nuclear fraction. As part of this previously unidentified complex,SNORD27 regulates the alternative splicing of the transcription factor E2F7p re-mRNA through direct RNA-RNA interaction without methylating the RNA, likely by competing with U1 small nuclear ribonucleoprotein (snRNP). Furthermore, knockdown of SNORD27 activates previously "silent" exons in several other genes through base complementarity across the entire SNORD27 sequence, not just the antisense boxes. Thus, some SNORDs likely function in both rRNA and pre-mRNA processing, which increases the repertoire of splicing regulators and links both processes.

Prader-Willi syndrome (PWS) is the predominant genetic cause of obesity in humans. Recent clinical reports have suggested that micro-deletion of the Snord116 gene cluster can lead to PWS, however, the extent of the contributions of the encoded snoRNAs is unknown. Here we show that mice lacking Snord116 globally have low birth weight, increased body weight gain, energy expenditure and hyperphagia. Consistent with this, microarray analysis of hypothalamic gene expression revealed a significant alteration in feeding related pathways that was also confirmed by in situ hybridisation. Importantly, selective deletion of Snord116 only from NPY expressing neurons mimics almost exactly the global deletion phenotype including the persistent low birth weight, increased body weight gain in early adulthood, increased energy expenditure and hyperphagia. Mechanistically, the lack of Snord116 in NPY neurons leads to the upregulation of NPY mRNA consistent with the hyperphagic phenotype and suggests a critical role of Snord116 in the control of NPY neuronal functions that might be dysregulated in PWS.

Small nucleolar RNAs (snoRNAs) are molecules located in the cell nucleolus and in Cajal bodies. Many scientific reports show that snoRNAs are not only responsible for modifications of other RNAs but also fulfill multiple other functions such as metabolic stress regulation or modulation of alternative splicing. Full-length snoRNAs as well as small RNAs derived from snoRNAs have been implied in human diseases such as cancer or Prader-Willi Syndrome. In this review we describe emerging, non-canonical roles of snoRNAs and their derivatives with the emphasis on their role in human diseases.

Non-coding RNAs (ncRNAs) have recently become increasingly important in the study of cellular metabolism and regulation such as development, proliferation, differentiation and apoptosis. However, the functions of most ncRNAs have remained largely unknown. Recently, studies have begun to characterize the aberrant regulation of ncRNAs in gastric cancer (GC) cells and tissues. These ncRNAs have a close relationship with drug resistance, and with the occurrence, development, invasion and metastasis of tumors, so they could possibly become new therapeutic targets and treatment tools for GC in the future. The present review summarized current advances in our knowledge of the roles of ncRNAs in GC., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Non-coding RNAs (ncRNAs) are important regulatory molecules involved in various physiological and pathological cellular processes. Small nucleolar RNAs (snoRNAs), subclass of small ncRNAs, have been considered important but unglamorous elements in the production of protein synthesis machinery of cells. However, recent evidence has indicated that these non-coding RNAs might have a crucial role also in controlling cell behavior, and snoRNAs dysfunction could significantly contribute to carcinogenesis. Here, we summarize the most important aspects of snoRNAs biology, their functioning in cancer cell, and potential usage in diagnosis or as a new class of therapeutic targets in cancer.

The majority of the eukaryotic genome is transcribed, generating a significant number of long intergenic noncoding RNAs (lincRNAs). Although lincRNAs represent the most poorly understood product of transcription, recent work has shown lincRNAs fulfill important cellular functions. In addition to low sequence conservation, poor understanding of structural mechanisms driving lincRNA biology hinders systematic prediction of their function. Here we report the molecular requirements for the recognition of steroid receptors (SRs) by the lincRNA growth arrest-specific 5 (Gas5), which regulates steroid-mediated transcriptional regulation, growth arrest and apoptosis. We identify the functional Gas5-SR interface and generate point mutations that ablate the SR-Gas5 lincRNA interaction, altering Gas5-driven apoptosis in cancer cell lines. Further, we find that the Gas5 SR-recognition sequence is conserved among haplorhines, with its evolutionary origin as a splice acceptor site. This study demonstrates that lincRNAs can recognize protein targets in a conserved, sequence-specific manner in order to affect critical cell functions.

We have employed gene-trap insertional mutagenesis to identify candidate genes whose disruption confer phenotypic resistance to lytic infection, in independent studies using 12 distinct viruses and several different cell lines. Analysis of >2,000 virus-resistant clones revealed >1,000 candidate host genes, approximately 20 % of which were disrupted in clones surviving separate infections with 2-6 viruses. Interestingly, there were 83 instances in which the insertional mutagenesis vector disrupted transcripts encoding H/ACA-class and C/D-class small nucleolar RNAs (SNORAs and SNORDs, respectively). Of these, 79 SNORAs and SNORDs reside within introns of 29 genes (predominantly protein-coding), while 4 appear to be independent transcription units. siRNA studies targeting candidate SNORA/Ds provided independent confirmation of their roles in infection when tested against cowpox virus, Dengue Fever virus, influenza A virus, human rhinovirus 16, herpes simplex virus 2, or respiratory syncytial virus. Significantly, eight of the nine SNORA/Ds targeted with siRNAs enhanced cellular resistance to multiple viruses suggesting widespread involvement of SNORA/Ds in virus-host interactions and/or virus-induced cell death.

Small nucleolar RNAs or snoRNAs, principally implicated in post-transcriptional chemical modification of other RNAs, were among the first non-coding RNA identified, together with ribosomal and transfer RNA. Lately, snoRNA have been involved in various unexpected functions, which renewed researcher's interest for these molecules. SnoRNA processing into smaller functional RNA species (sdRNA for snoRNA-derived RNA) or into miRNA (sno-miR), snoRNA mediated regulation of messenger RNA alternative splicing or snoRNA links to human disorders, including cancers, are some of the topics developed in this review., (© 2014 médecine/sciences – Inserm.)

Research into small non-coding RNAs (ncRNA) has fundamentally transformed our understanding of gene regulatory networks, especially at the post-transcriptional level. Although much is now known about the basic biology of small ncRNAs, our ability to recognize the impact of small ncRNA in disease states is preliminary, and the ability to effectively target them in vivo is very limited. However, given the larger and growing focus on targeting RNAs for disease therapeutics, what we do know about the intrinsic biology of these small RNAs makes them potentially attractive targets for pharmacologic manipulation. With that in mind, this review provides an introduction to the biology of small ncRNA, using microRNA (miRNA) and small nucleolar RNA (snoRNA) as examples.

Non-coding RNA GAS5 (growth arrest-specific transcript 5) is a 5'-TOP (5'-terminal oligopyrimidine tract) RNA, whose translation, and consequently also stability, is controlled by the mTOR (mammalian target of rapamycin) pathway. GAS5 was identified by functional expression cloning and is necessary and sufficient for normal growth arrest in both leukaemic and untransformed human T-lymphocytes. GAS5 is also required for the inhibitory effects of rapamycin and its analogues on T-cells. The striking functional effects of GAS5 may be mediated through the snoRNAs (small nucleolar RNAs) encoded in its introns and/or through the unusual folding of the mRNA itself, which sequesters, and therefore inhibits, the glucocorticoid receptor.

Background: To investigate small-nucleolar RNAs (snoRNAs) as reference genes when measuring miRNA expression in tumour samples, given emerging evidence for their role in cancer., Methods: Four snoRNAs, commonly used for normalisation, RNU44, RNU48, RNU43 and RNU6B, and miRNA known to be associated with pathological factors, were measured by real-time polymerase chain reaction in two patient series: 219 breast cancer and 46 head and neck squamous cell carcinoma (HNSCC). SnoRNA and miRNA were then correlated with clinicopathological features and prognosis., Results: Small-nucleolar RNA expression was as variable as miRNA expression (miR-21, miR-210, miR-10b). Normalising miRNA PCR expression data to these recommended snoRNAs introduced bias in associations between miRNA and pathology or outcome. Low snoRNA expression correlated with markers of aggressive pathology. Low levels of RNU44 were associated with a poor prognosis. RNU44 is an intronic gene in a cluster of highly conserved snoRNAs in the growth arrest specific 5 (GAS5) transcript, which is normally upregulated to arrest cell growth under stress. Low-tumour GAS5 expression was associated with a poor prognosis. RNU48 and RNU43 were also identified as intronic snoRNAs within genes that are dysregulated in cancer., Conclusion: Small-nucleolar RNAs are important in cancer prognosis, and their use as reference genes can introduce bias when determining miRNA expression.

Expression of dsRNA complementary to small nucleolar RNAs (snoRNAs) in Trypanosoma brucei results in snoRNA silencing, termed snoRNAi. Here, we demonstrate that snoRNAi requires the nuclear TbDCL2 protein, but not TbDCL1, which is involved in RNA interference (RNAi) in the cytoplasm. snoRNAi depends on Argonaute1 (Slicer), and on TbDCL2, suggesting that snoRNA dicing and slicing takes place in the nucleus, and further suggesting that AGO1 is active in nuclear silencing. snoRNAi was next utilized to elucidate the function of an abundant snoRNA, TB11Cs2C2 (92 nt), present in a cluster together with the spliced leader associated RNA (SLA1) and snR30, which are both H/ACA RNAs with special nuclear functions. Using AMT-UV cross-linking and RNaseH cleavage, we provide evidence for the interaction of TB11Cs2C2 with the small rRNAs, srRNA-2 and srRNA-6, which are part of the large subunit (LSU) rRNA. snoRNAi of TB11Cs2C2 resulted in defects in generating srRNA-2 and LSUβ rRNA. This is the first snoRNA described so far to engage in trypanosome-specific processing events.

The central importance of the serine/threonine protein kinase mTOR (mammalian Target of Rapamycin) in the control of cell growth and proliferation is well established. However, our knowledge both of the upstream pathways controlling mTOR activity and of the downstream events mediating these effects is still seriously incomplete. We report a previously unsuspected role for the nonprotein-coding RNA GAS5 in the inhibition of T-cell proliferation produced by mTOR antagonists such as rapamycin. GAS5 transcripts are up-regulated during growth arrest and after rapamycin treatment, and GAS5 has recently been shown to be necessary and sufficient for normal T-cell growth arrest. Down-regulation of GAS5 using RNA interference protects both leukemic and primary human T cells from the inhibition of proliferation produced by mTOR antagonists. The GAS5 transcript is a member of the 5' terminal oligopyrimidine class of RNAs, which is specifically controlled at the level of translation by the mTOR pathway, and the effects of GAS5 on the cell cycle provide a novel and important link to the control of proliferation. These observations point to a significant advance in our understanding of the mechanism of action of mTOR inhibitors, which is likely to lead to improvements in immunosuppressive and cancer therapy.

A significant portion of eukaryotic small ncRNA transcriptome is composed by small nucleolar RNAs. From archaeal to mammalian cells, these molecules act as guides in the site-specific pseudouridylation or methylation of target RNAs. We used a bioinformatics search program to detect Drosophila putative orthologues of U79, one out of ten snoRNAs produced by GAS5, a human ncRNA involved in apoptosis, susceptibility to cancer and autoimmune diseases. This search led to the definition of a list of U79-related fly snoRNAs whose genomic organization, evolution and expression strategy are discussed here. We report that an intriguing novel specimen, named Dm46E3, is transcribed as a longer, unspliced precursor from the reverse strand of eiger, a fly regulatory gene that plays a key role in cell differentiation, apoptosis and immune response. Expression of Dm46E3 was found significantly up-regulated in a mutant strain in which eiger transcription is greatly reduced, suggesting that these two sense-antisense genes may be mutually regulated. Relevant to its function, Dm46E3 concentrated specifically in the Cajal bodies, followed a dynamic spatial expression profile during embryogenesis and displayed a degenerate antisense element that enables it to target U1b, a developmentally regulated isoform of the U1 spliceosomal snRNA that is particularly abundant in embryos.

Retrovirus integrase (IN) integrates the viral linear DNA genome ( approximately 10 kb) into a host chromosome, a step which is essential for viral replication. Integration occurs via a nucleoprotein complex, termed the preintegration complex (PIC). This article focuses on the reconstitution of synaptic complexes from purified components whose molecular properties mirror those of the PIC, including the efficient concerted integration of two ends of linear viral DNA into target DNA. The methods described herein permit the biochemical and biophysical analyses of concerted integration. The methods enable (1) the study of interactions between purified recombinant IN and its viral DNA substrates at the molecular level; (2) the identification and characterization of nucleoprotein complexes involved in the human immunodeficiency virus type-1 (HIV-1) concerted integration pathway; (3) the determination of the multimeric state of IN within these complexes; (4) dissection of the interaction between HIV-1 IN and cellular proteins such as lens epithelium-derived growth factor (LEDGF/p75); (5) the examination of HIV-1 Class II and strand transfer inhibitor resistant IN mutants; (6) the mechanisms associated with strand transfer inhibitors directed against HIV-1 IN that have clinical relevance in the treatment of HIV-1/AIDS.

Effective control of both cell survival and cell proliferation is critical to the prevention of oncogenesis and to successful cancer therapy. Using functional expression cloning, we have identified GAS5 (growth arrest-specific transcript 5) as critical to the control of mammalian apoptosis and cell population growth. GAS5 transcripts are subject to complex post-transcriptional processing and some, but not all, GAS5 transcripts sensitize mammalian cells to apoptosis inducers. We have found that, in some cell lines, GAS5 expression induces growth arrest and apoptosis independently of other stimuli. GAS5 transcript levels were significantly reduced in breast cancer samples relative to adjacent unaffected normal breast epithelial tissues. The GAS5 gene has no significant protein-coding potential but expression encodes small nucleolar RNAs (snoRNAs) in its introns. Taken together with the recent demonstration of tumor suppressor characteristics in the related snoRNA U50, our observations suggest that such snoRNAs form a novel family of genes controlling oncogenesis and sensitivity to therapy in cancer.

The control of growth of lymphocyte populations is crucial to the physiological regulation of the immune system, and to the prevention of both leukaemic and autoimmune disease. This control is mediated through modulation of the cell cycle and regulation of cell death. During log-phase growth the rate of proliferation is high and there is a low rate of cell death. As the population density increases, the cell cycle is extended and apoptosis becomes more frequent as the population enters growth arrest. Here, we show that growth-arrest-specific transcript 5 (GAS5) plays an essential role in normal growth arrest in both T-cell lines and non-transformed lymphocytes. Overexpression of GAS5 causes both an increase in apoptosis and a reduction in the rate of progression through the cell-cycle. Consistent with this, downregulation of endogenous GAS5 inhibits apoptosis and maintains a more rapid cell cycle, indicating that GAS5 expression is both necessary and sufficient for normal growth arrest in T-cell lines as well as human peripheral blood T-cells. Control of apoptosis and the cell cycle by GAS5 has significant consequences for disease pathogenesis, because independent studies have already identified GAS5 as an important candidate gene in the development of autoimmune disease.

Small nucleolar RNA molecules (snoRNA) comprise a special kind of non-coding RNAs involved in the maturation process of rRNAs, snRNAs, tRNAs and mRNAs. Traditionally, these molecules have been divided into two families depending on the type of conserved boxes that they harbour: box C/D and H/ACA snoRNAs. Both types of snoRNAs are found associated with proteins forming a complex called snoRNP. Although some of the snoRNPs of each family mediate endonucleolytic cleavages of pre-rRNA, most of them participate in nucleotide modification: 2'-O- methylated nucleotides in the case of C/D snoRNPs and pseudouridine in the case of H/ACA snoRNPs. Based on published patents, the purpose of this review is to show the biotechnological impact of these molecules, which rely on their special features: participation in the functionality of ribosome, specific location on cell, and abnormal expression in some diseases like cancer.

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.

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 family of HEAT-repeat containing ribosome synthesis factors was previously identified in Saccharomyces cerevisiae. We report the detailed characterization of two of these factors, Utp10 and Utp20, which were initially identified as components of the small subunit processome. Coprecipitation analyses confirmed the association of Utp10 and Utp20 with U3 snoRNA and the early pre-rRNA processing intermediates. Particularly strong association was seen with aberrant processing intermediates, which may help target these RNAs for degradation. Genetic depletion of either protein inhibited the early pre-rRNA processing steps in 18S rRNA maturation but had little effect on pre-rRNA transcription or synthesis of the 25S or 5.8S rRNAs. The absence of the poly(A) polymerase Trf5, a component of the TRAMP5 complex and exosome cofactor, led to stabilization of the aberrant 23S RNA in strains depleted of Utp10 or Utp20. In the case of Utp10, 20S pre-rRNA synthesis was also modestly increased by this loss of surveillance activity.

The small nucleolar RNAs (snoRNAs) are associated with proteins in ribonucleoprotein complexes called snoRNPs ("snorps"). These complexes create modified nucleotides in preribosomal RNA and other RNAs and participate in nucleolytic cleavages of pre-rRNA. The various reactions occur in site-specific fashion, and the mature rRNAs are ultimately incorporated into cytoplasmic ribosomes. Most snoRNAs exist in two structural classes, and most members in each class are involved in nucleotide modification reactions. Guide snoRNAs in the "box C/D" class target methylation of the 2'-hydroxyl moiety, to form 2'-O-methylated nucleotides (Nm), whereas guide snoRNAs in the "box H/ACA" class target specific uridines for conversion to pseudouridine (Psi). The rRNA nucleotides modified in this manner are numerous, totaling approximately 100 in yeast and twice that number in humans. Although the chemistry of the modifications and the factors involved in their formation are largely explained, very little is known about the influence of the copious snoRNA-guided nucleotide modifications on rRNA activity and ribosome function. Among eukaryotic organisms the sites of rRNA modification and the corresponding guide snoRNAs have been best characterized in S. cerevisiae, making this a model organism for analyzing the consequences of modification. This chapter presents approaches to characterizing rRNA modification effects in yeast and includes strategies for evaluating a variety of specific rRNA functions. To aid in planning, a package of bioinformatics tools is described that enables investigators to correlate guide function with targeted ribosomal sites in several contexts. Genetic procedures are presented for depleting modifications at one or more rRNA sites, including ablation of all Nm or Psi modifications made by snoRNPs, and for introducing modifications at novel sites. Methods are also included for characterizing modification effects on cell growth, antibiotic sensitivity, rRNA processing, formation of various rRNP complexes, translation activity, and rRNA structure within the ribosome.

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 http://gene.fudan.sh.cn/snoRNAbase.nsf.

The nucleolus is the largest and most dynamic nuclear domain in the vast majority of eukaryotic cells. The main and universal nucleolar function is participation in ribosome biogenesis, including ribosomal DNA (rDNA) transcription, pre-rRNA processing and ribosome subunit assembly. Furthermore, the nucleolus and its proteins also participate in cell cycle regulation, apoptosis and cell aging. These nucleolar functions are realized predominantly in interphase and, apparently, are abolished during mitosis, when the nucleolus disassembles. In this review, literature and our own data on the dynamics and mechanisms of the nucleolus disassembly and reassembly during mitosis in animal and plant cells are summarized. Particular attention is paid to the results obtained by analysis of the nucleolar dynamics in living cells and to modeling of the premature assembly of nucleolus upon various experimental conditions.

For a long time, molecular evolutionary biologists have been focused on DNA and proteins, whereas RNA has lived in the shadow of its famous chemical cousins as a mere intermediary. Although this perspective has begun to change since genome-wide transcriptional profiling was successfully extended to evolutionary biology, it still echoes in evolutionary literature. In this mini-review, new developments of RNA biochemistry and transcriptomics are brought to the attention of evolutionary biologists. In particular, the unexpected abundance and functional significance of noncoding RNAs is briefly reviewed. Noncoding RNAs control a remarkable range of biological pathways and processes, all with obvious fitness consequences, such as initiation of translation, mRNA abundance, transposon jumping, chromosome architecture, stem cell maintenance, development of brain and muscles, insulin secretion, cancerogenesis and plant resistance to viral infections.

Increasing evidence suggests that the development and function of the nervous system is heavily dependent on RNA editing and the intricate spatiotemporal expression of a wide repertoire of non-coding RNAs, including micro RNAs, small nucleolar RNAs and longer non-coding RNAs. Non-coding RNAs may provide the key to understanding the multi-tiered links between neural development, nervous system function, and neurological diseases.

The nucleolus is the most prominent compartment in the nucleus and known as the site for ribosome biogenesis in eucaryotes. In contrast, there is no such equivalent structure for ribosome synthesis in procaryotes. This raises two concerns that how does the nucleolus evolve and that whether the nucleolus remains playing a single role in ribosome biogenesis along the evolution. Increasing data support new nucleolus functions, including signal recognition particle assembly, small RNA modification, telomerase maturation, cell-cycle and aging control, and cell stress sensor. Multiple functions of the nucleolus possibly result from the plurifunctionality of nucleolar proteins, such as nucleolin and Nopp140. Proteomic analyses of human and Arabidopsis nucleolus lead a remarkable progress in understanding the evolution and new functions of nucleoli. In this review, we present a brief history of nucleolus research and new concepts and unresolved questions. Also, we introduce hepatitis D virus for studying the communication between the nucleolus and other subnuclear compartments, and Caenorhabditis elegans for the role of nucleolus in the development and the epistatic control of nucleologenesis.

The Prader-Willi syndrome is a congenital disease that is caused by the loss of paternal gene expression from a maternally imprinted region on chromosome 15. This region contains a small nucleolar RNA (snoRNA), HBII-52, that exhibits sequence complementarity to the alternatively spliced exon Vb of the serotonin receptor 5-HT(2C)R. We found that HBII-52 regulates alternative splicing of 5-HT(2C)R by binding to a silencing element in exon Vb. Prader-Willi syndrome patients do not express HBII-52. They have different 5-HT(2C)R messenger RNA (mRNA) isoforms than healthy individuals. Our results show that a snoRNA regulates the processing of an mRNA expressed from a gene located on a different chromosome, and the results indicate that a defect in pre-mRNA processing contributes to the Prader-Willi syndrome.

The snoRNA-LBME-db is a dedicated database containing human C/D box and H/ACA box small nucleolar RNAs (snoRNAs), and small Cajal body-specific RNAs (scaRNAs). C/D box and H/ACA box snoRNAs are part of ribonucleoparticles that guide 2'-O-ribose methylation and pseudouridilation, respectively, of selected residues of 28S, 18S or 5.8S rRNAs or of the spliceosomal U6 RNA. Similarly, scaRNAs guide modifications of the spliceosomal RNAs transcribed by RNA polymerase II (U1, U2, U4, U5 and U12) and are often composed of both C/D box and H/ACA box domains. However, some snoRNAs do not function as modification guide RNAs, but rather as RNA chaperones during the maturation of pre-rRNA. The database was built by a compilation of the literature, and comprises human sno/scaRNAs that were experimentally verified, as well as the human orthologs of snoRNAs that were cloned in other vertebrate species, and some snoRNAs that are predicted by bioinformatics search in loci submitted to genomic imprinting, but have not all been experimentally verified. For each entry, the database identifies the modified nucleotide(s) in the target RNA(s), indicates the corresponding predicted base pairing, gives a few pertinent references and provides a link to the position of the sno/scaRNA on the UCSC Genome Browser. The 'Find guide RNA' function allows one to find the sno/scaRNAs predicted to guide the modification of a particular nucleotide in the rRNA and spliceosomal RNA sequences. The 'Browse' function allows one to download the sequences of selected sno/scaRNAs in the FASTA format. The database is available online at http://www-snorna.biotoul.fr/. It can also be accessed from the human UCSC Genome Browser via the sno/miRNA track.

One of the largest families of small RNAs in eukaryotes is the H/ACA small nucleolar RNAs (snoRNAs), most of which guide RNA pseudouridine formation. So far, an effective computational method specifically for identifying H/ACA snoRNA gene sequences has not been established. We have developed snoGPS, a program for computationally screening genomic sequences for H/ACA guide snoRNAs. The program implements a deterministic screening algorithm combined with a probabilistic model to score gene candidates. We report here the results of testing snoGPS on the budding yeast Saccharomyces cerevisiae. Six candidate snoRNAs were verified as novel RNA transcripts, and five of these were verified as guides for pseudouridine formation at specific sites in ribosomal RNA. We also predicted 14 new base-pairings between snoRNAs and known pseudouridine sites in S.cerevisiae rRNA, 12 of which were verified by gene disruption and loss of the cognate pseudouridine site. Our findings include the first prediction and verification of snoRNAs that guide pseudouridine modification at more than two sites. With this work, 41 of the 44 known pseudouridine modifications in S.cerevisiae rRNA have been linked with a verified snoRNA, providing the most complete accounting of the H/ACA snoRNAs that guide pseudouridylation in any species.

Small non-messenger RNAs (snmRNAs) are a heterogeneous group of non-coding RNAs with a variety of regulatory functions including regulation of protein expression and guidance in RNA modifications. They are actively being investigated in Archaebacteria, yeast, invertebrates and mammals. Brain-specific snmRNAs have been identified in mammals and they seem to contribute to neuronal differentiation during development and to brain functions subserving learning and memory. Here we review the current knowledge of the properties, expression and functions of three groups of brain-specific snmRNAs: small nucleolar RNAs, BC1/BC200 RNAs and microRNAs.

Through a computer search of DNA databases, we have identified the homologs of the mgU6-47 snoRNA gene from the yeast Schizosaccharomyces pombe, the fly Drosophila melanogaster and human. The three box C/D-containing snoRNA genes showed no significant similarity in their sequences except for an 11 nt long complementarity to U6 snRNA, suggesting that the mechanism of snoRNA guided snRNA methylation is conserved from mammals to yeast. The corresponding snoRNAs have been positively detected by reverse transcription and northern blotting. Taking advantage of the fission yeast system, we have disrupted the yeast mgU6-47 gene and demonstrated that it is absolutely required for site-specific 2'-O-methylation of U6 at position A41. No growth differences between mgU6-47 gene-disrupted and wild-type cells were observed, suggesting that the mgU6-47 gene, as for most rRNA methylation guides, is dispensable in yeast. Nevertheless, it was revealed by temperature shift assay that abolition of A41 methylation in yeast U6 snRNA might cause a small decrease in mRNA splicing efficiency. The timing of S.pombe U6 pre-RNA transport in the nucleus for splicing and methylation was also analyzed and is described.

The small nucleolar RNAs (snoRNAs) are an abundant class of trans-acting RNAs that function in ribosome biogenesis in the eukaryotic nucleolus. Elegant work has revealed that most known snoRNAs guide modification of pre-ribosomal RNA (pre-rRNA) by base pairing near target sites. Other snoRNAs are involved in cleavage of pre-rRNA by mechanisms that have not yet been detailed. Moreover, our appreciation of the cellular roles of the snoRNAs is expanding with new evidence that snoRNAs also target modification of small nuclear RNAs and messenger RNAs. Many snoRNAs are produced by unorthodox modes of biogenesis including salvage from introns of pre-mRNAs. The recent discovery that homologs of snoRNAs as well as associated proteins exist in the domain Archaea indicates that the RNA-guided RNA modification system is of ancient evolutionary origin. In addition, it has become clear that the RNA component of vertebrate telomerase (an enzyme implicated in cancer and cellular senescence) is related to snoRNAs. During its evolution, vertebrate telomerase RNA appears to have co-opted a snoRNA domain that is essential for the function of telomerase RNA in vivo. The unique properties of snoRNAs are now being harnessed for basic research and therapeutic applications.

Several snoRNAs are essential for the sequence of cleavage events required to produce the mature forms of 18S, 5.8S, and 28S rRNA from the large precursor molecule. In the absence of U22, mature 18S rRNA fails to accumulate; U8 snoRNA is essential for accumulation of both 5.8S and 28S rRNA. The mechanisms by which snoRNAs facilitate these cleavage events is not known and might include direct cleavage or assisting the rate or efficiency of ribosome assembly. To learn more about the mechanisms of snoRNA-mediated pre-rRNA processing, an examination of the kinetics of pre-rRNA processing in Xenopus oocytes was undertaken. Correct pre-rRNA processing can be restored in snoRNA-depleted oocytes following cytoplasmic injection of the corresponding in vitro-synthesized snoRNA. Analysis of the kinetics of pre-rRNA processing in these snoRNA-rescue experiments demonstrated that the rate of accumulation of mature rRNAs was slower than that seen in untreated oocytes. The snoRNAs were imported into the nucleus at a rate and overall efficiency less than that of U1 snRNA, used as a control for import. However, sufficient levels of snoRNA were present in the nucleus to yield a functional phenotype (rescue of rRNA processing) several hours before the snoRNAs were directly detectable in the nucleus via autoradiography. This indicated that very low amounts of the snoRNA in the nucleus were sufficient for rescue. Finally, transcriptional inhibitors were used to separate transcription and processing. Failure to rescue snoRNA-mediated processing of pre-accumulated precursors is consistent with a scenario in which U8 and U22 must be present during transcription of pre-rRNA.

The eukaryotic nucleolus contains a diverse population of small nucleolar RNAs (snoRNAs) essential for ribosome biogenesis. The box C/D snoRNA family possesses conserved nucleotide boxes C and D that are multifunctional elements required for snoRNA processing, snoRNA transport to the nucleolus, and 2'-O-methylation of ribosomal RNA. We have previously demonstrated that the assembly of an snoRNP complex is essential for processing the intronic box C/D snoRNAs and that specific nuclear proteins associate with the box C/D core motif in vitro. Using a box C/D motif derived from mouse U14 snoRNA, we have now affinity purified and defined four mouse proteins that associate with this minimal RNA substrate. These four proteins consist of two protein pairs: members of each pair are highly related in sequence. One protein pair corresponds to the essential yeast nucleolar proteins Nop56p and Nop58p. Affinity purification of mouse Nop58 confirms observations made in yeast that Nop58 is a core protein of the box C/D snoRNP complex. Isolation of Nop56 using this RNA motif defines an additional snoRNP core protein. The second pair of mouse proteins, designated p50 and p55, are also highly conserved among eukaryotes. Antibody probing of nuclear fractions revealed a predominance of p55 and p50 in the nucleoplasm, suggesting a possible role for the p50/p55 pair in snoRNA production and/or nucleolar transport. The reported interaction of p55 with TATA-binding protein (TBP) and replication A protein as well as the DNA helicase activity of p55 and p50 may suggest the coordination of snoRNA processing and snoRNP assembly with replication and/or transcriptional events in the nucleus. Homologs for both snoRNA-associated protein pairs occur in Archaea, strengthening the hypothesis that the box C/D RNA elements and their interacting proteins are of ancient evolutionary origin.

Many small RNA species associate with the nucleolar structure. Some of these small nucleolar RNAs (snoRNAs) are required for cleavage processing of ribosomal RNA precursors. There are many pseudouridine residues and methylated riboses in mature ribosomal RNA. For most, if not all, of these modifications, each site is selected by base pairing with a specific snoRNA species. Some snoRNAs are needed for the 2'-O-ribose methylation of at least one spliceosomal small nuclear RNA. Many snoRNAs, particularly in yeast, are generated from independent transcription units. Most vertebrate snoRNAs are produced by processing of introns from protein-coding transcripts. Some snoRNAs are made by processing of introns from non-protein-coding transcripts.