The effect of chromosome abnormalities on expression of SnoRNA in radioresistant and radiosensitive cell lines after irradiation.

In this paper, we have studied the role of chromosomal abnormalities in the expression of small nucleolar RNAs (snoRNAs) of radioresistant (K562) and radiosensitive (HL-60) leukemia cell line. Cells were exposed to an X-ray dose of 4 Gy. SnoRNA expression was investigated using NGS sequencing. The distribution of expressed snoRNAs on chromosomes has been found to be different for two cell lines. The most significant differences in the expression of snoRNAs were found in the K562 cell line based on the analysis of the dynamics of log2fc values. The type of clustering, the number and type of snoRNAs slightly differed in the chromosomes with trisomy and monosomy and had a pronounced difference in pairs with marker chromosomes in both cell lines. In this study, we have demonstrated that chromosomal abnormalities alter the expression of snoRNA after irradiation. Trisomies and monosomies do not have such a noticeable effect on the expression of snoRNAs as the presence of marker chromosomes.

Revealing the clinical significance and prognostic value of small nucleolar RNA SNORD31 in hepatocellular carcinoma.

BACKGROUND: For lack of accurate early diagnosis and prognostic assessment, hepatocellular carcinoma (HCC) becomes severe challenge with the fourth cancer-related mortality. Recently, non-coding RNA (ncRNA) was identified to make functions in progression of various tumors. Among that, a novel ncRNA, small nucleolar RNA C/D box 31 (SNORD31) was suggested in previous study to function as potential tumor suppressing role. In the present study, we aimed to investigate the expression patterns and clinical significance of SNORD31 in HCC. METHODS: SNORD31 expression was calculated in HCC cell lines as well as clinical specimens by RT-PCR. HCC patients were subdivided into high and low SNORD31 expression groups and their clinical characteristics were compared. Besides, the association between SNORD31 expression and postoperative prognosis was evaluated using Kaplan-Meier and Cox regression analysis. RESULTS: Compared with corresponding normal reference, expression levels of SNORD31 were significantly down-regulated in both HCC cell lines and clinical specimens (P<0.01). Moreover, low SNORD31 expression was remarkably correlated with large tumor diameter, high incidence of vessel carcinoma embolus and capsular invasion, severe tumor differentiation and tumor-node-metastasis (TNM) stage (P<0.05). In the following analysis, HCC patients with low SNORD31 expression were independently inclined with poor tumor-free (median time: 9.17 vs 48.8 months, low vs high, P<0.001) as well as long-term survival (LTS; median time: 40.26 vs 55.41 months, low vs high, P=0.002). CONCLUSIONS: The ncRNA SNORD31 was proved to be commonly down-regulated in HCC and was independently associated with multiple malignant characteristics and long-term prognosis of HCC patients, which implied that SNORD31 possessed potential as a novel HCC biomarker.

Small Nucleolar RNA Score: An Assay to Detect Formalin-Overfixed Tissue.

Although there are millions of formalin-fixed paraffin-embedded (FFPE) tissue blocks potentially available for scientific research, many are of questionable quality, partly due to unknown fixation conditions. We analyzed FFPE tissue biospecimens as part of the NCI Biospecimen Preanalytical Variables (BPV) program to identify microRNA (miRNA) markers for fixation time. miRNA was extracted from kidney and ovary tumor FFPE blocks (19 patients, cold ischemia ≤2 hours) with 6, 12, 24, and 72 hours fixation times, then analyzed using the WaferGen SmartChip platform (miRNA chip with 1036 miRNA targets). For fixation time, principal component analysis of miRNA chip expression data separated 72 hours fixed samples from 6 to 24 hours fixed samples. A set of small nuclear RNA (snRNA) targets was identified that best determines fixation time and was validated using a second independent cohort of seven different tissue types. A customized assay was then developed, based on a set of 24 miRNA and snRNA targets, and a simple "snoRNA score" defined. This score detects FFPE tissue samples with fixation for 72 hours or more, with 79% sensitivity and 80% specificity. It can therefore be used to assess the fitness-for-purpose of FFPE samples for DNA or RNA-based research or clinical assays, which are known to be of limited robustness to formalin overfixation.

Live-cell imaging of small nucleolar RNA tagged with the broccoli aptamer in yeast.

The development of the RNA 'vegetable' aptamers, Spinach and Broccoli, has simplified RNA imaging, especially in live cells. These RNA aptamers interact with a fluorophore (DFHBI or DFHBI-1T) to produce a green fluorescence signal. Although used in mammalian and Escherichia coli cells, the use of these aptamers in yeast has been limited. Here we describe how the Saccharomyces cerevisiae snoRNA, snR30, was tagged with the Spinach or the Broccoli aptamers and observed in live cells. The ability to observe aptamer fluorescence in polyacrylamide gels stained with a fluorophore or with a microplate reader can ease preliminary screening of the aptamers in different RNA scaffolds. In snR30 a tandem repeat of the Broccoli aptamer produced the best signal in vitro. Multiple factors in cell preparation were vital for obtaining a good fluorescence signal. These factors included the clearance of the native unmodified snR30, the amount and length of dye incubation and the rinsing of cells. In cells, the aptamers did not interfere with the structure or essential function of snR30, as the tagged RNA localized to the nucleolus and directed processing of ribosomal RNA in yeast. High-resolution images of the tagged snoRNA were obtained with live cells immobilized by a microcompressor.

Expression profiling of snoRNAs in normal hematopoiesis and AML.

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that contribute to ribosome biogenesis and RNA splicing by modifying ribosomal RNA and spliceosome RNAs, respectively. We optimized a next-generation sequencing approach and a custom analysis pipeline to identify and quantify expression of snoRNAs in acute myeloid leukemia (AML) and normal hematopoietic cell populations. We show that snoRNAs are expressed in a lineage- and development-specific fashion during hematopoiesis. The most striking examples involve snoRNAs located in 2 imprinted loci, which are highly expressed in hematopoietic progenitors and downregulated during myeloid differentiation. Although most snoRNAs are expressed at similar levels in AML cells compared with CD34+, a subset of snoRNAs showed consistent differential expression, with the great majority of these being decreased in the AML samples. Analysis of host gene expression, splicing patterns, and whole-genome sequence data for mutational events did not identify transcriptional patterns or genetic alterations that account for these expression differences. These data provide a comprehensive analysis of the snoRNA transcriptome in normal and leukemic cells and should be helpful in the design of studies to define the contribution of snoRNAs to normal and malignant hematopoiesis.

Small Nucleolar Noncoding RNA SNORA23, Up-Regulated in Human Pancreatic Ductal Adenocarcinoma, Regulates Expression of Spectrin Repeat-Containing Nuclear Envelope 2 to Promote Growth and Metastasis of Xenograft Tumors in Mice.

BACKGROUND & AIMS: Small nucleolar noncoding RNAs (snoRNAs) regulate function of ribosomes, and specific snoRNAs are dysregulated in some cancer cells. We investigated dysregulation of snoRNAs in pancreatic ductal adenocarcinoma (PDAC) cells. METHODS: We investigated snoRNA expression in PDAC cell lines by complementary DNA microarray and quantitative reverse transcription polymerase chain reaction. In PDAC (n = 133), intraductal papillary mucinous neoplasm (n = 16), mucinous cystic neoplasm-associated PDAC (n = 1), and non-tumor pancreas (n = 8) and liver (n = 3) tissues from subjects who underwent surgical resection, levels of snoRNA were measured by quantitative reverse transcription polymerase chain reaction and compared with clinicopathologic parameters and survival times determined by Kaplan-Meier analysis. To examine snoRNA function, PDAC cells were transfected with snoRNA-antisense oligonucleotides flanked with amido-bridged nucleic acids, or snoRNA-expression plasmids, and analyzed in proliferation, colony formation, spheroid formation, and invasion assays. To identify snoRNA-related factors, cells were analyzed by gene expression and proteomic profiling and immunoblot assays. Mice were given intrasplenic injections of MIA PaCa2- or Suit2-HLMC cells; tumor-bearing nude mice were then given 3 weekly injections of an antisense oligonucleotides against SNORA23, a H/ACA-box type snoRNA, and tumor growth and metastasis to liver, blood, and pancreas were analyzed. RESULTS: Levels of SNORA23 increased and accumulated at the nucleolus in highly metastatic MIA PaCa2- or Suit2-HLMC cells compared with their parental cells. We detected SNORA23 in human PDAC specimens but not in non-tumor pancreatic tissue. PDAC level of SNORA23 correlated with invasion grade and correlated inversely with disease-free survival time of patients. Expression of SNORA23 in PDAC cells increased their invasive activity and colony formation, and spheroid formation was inhibited by SNORA23 knockdown. In gene expression and proteomic profile analyses, we found SNORA23 to increase expression of spectrin repeat-containing nuclear envelope 2 (SYNE2) messenger RNA and protein. Knockdown of SYNE2 in PDAC cells reduced their invasive activities and anchor-independent survival. Administration of SNORA23 antisense oligonucleotides to mice slowed growth of xenograft tumors, tumor expression of SYNE2, tumor cell dissemination, and metastasis to liver. CONCLUSIONS: We found expression of the snoRNA SNORA23, which mediates sequence-specific pseudouridylation of ribosomal RNAs, to be increased in human PDAC tissues compared with non-tumor tissues, and levels to correlate with tumor invasion grade and patient survival time. SNORA23 increases expression of SYNE2, possibly through modulation of ribosome biogenesis, to promote PDAC cell survival and invasion, and growth and metastasis of xenograft tumors in mice.

A micro-RNA expression signature for human NAFLD progression.

BACKGROUND: The spectrum of nonalcoholic fatty liver disease (NAFLD) describes disease conditions deteriorating from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) to cirrhosis (CIR) to hepatocellular carcinoma (HCC). From a molecular and biochemical perspective, our understanding of the etiology of this disease is limited by the broad spectrum of disease presentations, the lack of a thorough understanding of the factors contributing to disease susceptibility, and ethical concerns related to repeat sampling of the liver. To better understand the factors associated with disease progression, we investigated by next-generation RNA sequencing the altered expression of microRNAs (miRNAs) in liver biopsies of class III obese subjects (body mass index ≥40 kg/m(2)) biopsied at the time of elective bariatric surgery. METHODS: Clinical characteristics and unbiased RNA expression profiles for 233 miRs, 313 transfer RNAs (tRNAs), and 392 miscellaneous small RNAs (snoRNAs, snRNAs, rRNAs) were compared among 36 liver biopsy specimens stratified by disease severity. RESULTS: The abundances of 3 miRNAs that were found to be differentially regulated (miR-301a-3p and miR-34a-5p increased and miR-375 decreased) with disease progression were validated by RT-PCR. No tRNAs or miscellaneous RNAs were found to be associated with disease severity. Similar patterns of increased miR-301a and decreased miR-375 expression were observed in 134 hepatocellular carcinoma (HCC) samples deposited in The Cancer Genome Atlas (TCGA). CONCLUSIONS: Our analytical results suggest that NAFLD severity is associated with a specific pattern of altered hepatic microRNA expression that may drive the hallmark of this disorder: altered lipid and carbohydrate metabolism. The three identified miRNAs can potentially be used as biomarkers to access the severity of NAFLD. The persistence of this miRNA expression pattern in an external validation cohort of HCC samples suggests that specific microRNA expression patterns may permit and/or sustain NAFLD development to HCC.

ECHO-liveFISH: in vivo RNA labeling reveals dynamic regulation of nuclear RNA foci in living tissues.

Elucidating the dynamic organization of nuclear RNA foci is important for understanding and manipulating these functional sites of gene expression in both physiological and pathological states. However, such studies have been difficult to establish in vivo as a result of the absence of suitable RNA imaging methods. Here, we describe a high-resolution fluorescence RNA imaging method, ECHO-liveFISH, to label endogenous nuclear RNA in living mice and chicks. Upon in vivo electroporation, exciton-controlled sequence-specific oligonucleotide probes revealed focally concentrated endogenous 28S rRNA and U3 snoRNA at nucleoli and poly(A) RNA at nuclear speckles. Time-lapse imaging reveals steady-state stability of these RNA foci and dynamic dissipation of 28S rRNA concentrations upon polymerase I inhibition in native brain tissue. Confirming the validity of this technique in a physiological context, the in vivo RNA labeling did not interfere with the function of target RNA nor cause noticeable cytotoxicity or perturbation of cellular behavior.

Genome-wide small nucleolar RNA expression analysis of lung cancer by next-generation deep sequencing.

Emerging evidence indicates that small nucleolar RNAs (snoRNAs), a class of small noncoding RNAs, may play important function in tumorigenesis. Nonsmall-cell lung cancer (NSCLC) is the number one cancer killer for men and women. Systematically characterizing snoRNAs in NSCLC will develop biomarkers for its early detection and prognostication. We used next-generation deep sequencing to comprehensively characterize snoRNA profiles in 12 NSCLC tissues. We used quantitative reverse transcription polymerase chain reaction (qRT-PCR) to verify the findings in 40 surgical Stage I NSCLC specimens and 126 frozen NSCLC tissues of different stages. The 126 NSCLC tissues were divided into a training set and a testing set. Deep sequencing identified 458 snoRNAs, of which, 29 had a ≥3.0-fold expression level change in Stage I NSCLC tissues versus normal tissues. qRT-PCR analysis showed that 16 of 29 snoRNAs exhibited consistent changes with deep sequencing data. The 16 snoRNAs exhibited 0.75-0.94 area under receiver-operator characteristic curve values in distinguishing lung tumor from normal lung tissues (all ≤0.0001) with 70.0-95.0% sensitivity and 70.0-95.0% specificity. Six genes (snoRA47, snoRA68, snoRA78, snoRA21, snoRD28 and snoRD66) were identified whose expressions were associated with overall survival of the NSCLC patients. A prediction model consisting of three genes (snoRA47, snoRA68 and snoRA78) was developed in the training set of 77 cases, which could significantly predict overall survival of the NSCLC patients (p < 0.0001). The prognostic performance of the prediction model was confirmed in the testing set of 49 NSCLC patients. The identified snoRNA signatures may provide potential biomarkers for the early detection and prognostication of NSCLC.

Small nucleolar RNA host genes and long non-coding RNA responses in directly irradiated and bystander cells.

The irradiated cells communicate with unirradiated cells and induce changes in them through a phenomenon known as the bystander effect. The nature of the bystander signal and how it impacts unirradiated cells remains to be discovered. Examination of molecular changes could lead to the identification of pathways underlying the bystander effect. Apart from microRNAs, little is known about the regulation of other non-coding RNAs (ncRNA) in irradiated or bystander cells. In this study we monitored the transcriptional changes of several small nucleolar RNAs (snoRNAs) host genes and long non-coding RNAs (lncRNAs) that are known to participate in a variety of cellular functions, in irradiated and bystander cells to gain insight into the molecular pathways affected in these cells. We used human lymphoblasts TK6 cells in a medium exchanged bystander effect model system to examine ncRNA expression alterations. The snoRNA host genes SNHG1 and SNHG4 were upregulated in irradiated TK6 cells but were repressed in bystander cells. The SNHG5 and SNHG11 were downregulated in irradiated and bystander cells and the expression levels of these ncRNA were significantly lower in bystander cells. The lncRNA MALAT1, MATR3, SRA1, and SOX2OT were induced in irradiated TK6 cells and their expression levels were repressed in bystander cells. The lncRNA RMST was induced in both irradiated and bystander cells. Taken together, these results indicate that expression levels of ncRNA are modulated in irradiated and bystander cells and these transcriptional changes could be associated with the bystander effect.

Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core proteins and small RNA sequencing.

BACKGROUND: In recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequence long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated proteins. RESULTS: Analysis of these data sets reveals that many loci in the human genome reproducibly give rise to C/D box-like snoRNAs, whose expression and evolutionary conservation are typically less pronounced relative to the snoRNAs that are currently cataloged. We further find that virtually all C/D box snoRNAs are specifically processed inside the regions of terminal complementarity, retaining in the mature form only 4-5 nucleotides upstream of the C box and 2-5 nucleotides downstream of the D box. Sequencing of the total and Argonaute 2-associated populations of small RNAs reveals that despite their cellular abundance, C/D box-derived small RNAs are not efficiently incorporated into the Ago2 protein. CONCLUSIONS: We conclude that the human genome encodes a large number of snoRNAs that are processed along the canonical pathway and expressed at relatively low levels. Generation of snoRNA-derived processing products with alternative, particularly miRNA-like, functions appears to be uncommon.

The small-nucleolar RNAs commonly used for microRNA normalisation correlate with tumour pathology and prognosis.

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.

SnoRNA microarray analysis reveals changes in H/ACA and C/D RNA levels caused by dyskerin ablation in mouse liver.

snoRNAs (small nucleolar RNAs) are key components of snoRNP (small nucleolar ribonucleoprotein) particles involved in modifying specific residues of ribosomal and other RNAs by pseudouridylation (H/ACA snoRNAs) or methylation (C/D snoRNAs). They are encoded within the introns of host genes, which tend to be genes whose products are involved in ribosome biogenesis or function. Although snoRNPs are abundant, ubiquitous and their components highly conserved, information concerning their expression during development or how their expression is altered in diseased states is sparse. To facilitate these studies we have developed a snoRNA microarray platform for the analysis of the abundance of snoRNAs in different RNA samples. In the present study we show that the microarray is sensitive and specific for the detection of snoRNAs. A mouse snoRNA microarray was used to monitor changes in abundance of snoRNAs after ablation of dyskerin, an H/ACA snoRNA protein component, from mouse liver, which causes a decrease in ribosome production. H/ACA snoRNAs were decreased in abundance in these livers while, unexpectedly, C/D snoRNAs were increased. The increase in C/D snoRNAs corresponded with an increase in the abundance of the mRNAs transcribed from snoRNA host genes, suggesting the increase may be part of a cellular response to defective ribosome synthesis.

SnoPatrol: how many snoRNA genes are there?

Small nucleolar RNAs (snoRNAs) are among the most evolutionarily ancient classes of small RNA. Two experimental screens published in BMC Genomics expand the eukaryotic snoRNA catalog, but many more snoRNAs remain to be found.

Mining small RNA sequencing data: a new approach to identify small nucleolar RNAs in Arabidopsis.

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that direct 2'-O-methylation or pseudouridylation on ribosomal RNAs or spliceosomal small nuclear RNAs. These modifications are needed to modulate the activity of ribosomes and spliceosomes. A comprehensive repertoire of snoRNAs is needed to expand the knowledge of these modifications. The sequences corresponding to snoRNAs in 18-26-nt small RNA sequencing data have been rarely explored and remain as a hidden treasure for snoRNA annotation. Here, we showed the enrichment of small RNAs at Arabidopsis snoRNA termini and developed a computational approach to identify snoRNAs on the basis of this characteristic. The approach successfully uncovered the full-length sequences of 144 known Arabidopsis snoRNA genes, including some snoRNAs with improved 5'- or 3'-end annotation. In addition, we identified 27 and 17 candidates for novel box C/D and box H/ACA snoRNAs, respectively. Northern blot analysis and sequencing data from parallel analysis of RNA ends confirmed the expression and the termini of the newly predicted snoRNAs. Our study especially expanded on the current knowledge of box H/ACA snoRNAs and snoRNA species targeting snRNAs. In this study, we demonstrated that the use of small RNA sequencing data can increase the complexity and the accuracy of snoRNA annotation.

NLS peptide conjugated molecular beacons for visualizing nuclear RNA in living cells.

Imaging the expression and localization of RNAs in live-cell nucleus can provide important information on RNA synthesis, processing, and transport. Here, we report the development of a bifunctional molecular beacon (NLS-MB) composed of a single nuclear localization sequence (NLS) peptide conjugated to a molecular beacon for efficient delivery and imaging of endogenous RNAs in the nuclei of living cells. We characterized the NLS-MBs by comparing their signal-to-noise ratios with unmodified molecular beacons and determined their efficiency of nuclear import. We demonstrated the specificity and sensitivity of the method by observing in living cells the localization and colocalization of small nuclear RNAs (snRNA) U1 and U2 at discrete foci in the nucleoplasm, and the localization of small nucleolar RNA U3 in the nucleolus. These snRNAs were chosen because of their essential roles in RNA biogenesis. The results were validated using in situ hybridization as positive control and random beacons as negative control. This novel approach may be applied to imaging other nuclear RNAs and pre-mRNAs in living cells.

Identification of genes that function in the biogenesis and localization of small nucleolar RNAs in Saccharomyces cerevisiae.

Small nucleolar RNAs (snoRNAs) orchestrate the modification and cleavage of pre-rRNA and are essential for ribosome biogenesis. Recent data suggest that after nucleoplasmic synthesis, snoRNAs transiently localize to the Cajal body (in plant and animal cells) or the homologous nucleolar body (in budding yeast) for maturation and assembly into snoRNPs prior to accumulation in their primary functional site, the nucleolus. However, little is known about the trans-acting factors important for the intranuclear trafficking and nucleolar localization of snoRNAs. Here, we describe a large-scale genetic screen to identify proteins important for snoRNA transport in Saccharomyces cerevisiae. We performed fluorescence in situ hybridization analysis to visualize U3 snoRNA localization in a collection of temperature-sensitive yeast mutants. We have identified Nop4, Prp21, Tao3, Sec14, and Htl1 as proteins important for the proper localization of U3 snoRNA. Mutations in genes encoding these proteins lead to specific defects in the targeting or retention of the snoRNA to either the nucleolar body or the nucleolus. Additional characterization of the mutants revealed impairment in specific steps of U3 snoRNA processing, demonstrating that snoRNA maturation and trafficking are linked processes.

Distribution of U3 small nucleolar RNA and fibrillarin during early embryogenesis in Caenorhabditis elegans.

U3 small nucleolar RNA (snoRNA) is one of the members of the box C/D class of snoRNA and is essential for ribosomal RNA (rRNA) processing to generate 18S rRNA in the nucleolus. Although U3 snoRNA is abundant, and is well conserved from yeast to mammals, the genes encoding U3 snoRNA in C. elegans have long remained unidentified. A recent RNomics study in C. elegans predicted five distinct U3 snoRNA genes. However, characterization of these candidates for U3 snoRNA has yet to be performed. In this study, we isolated and characterized four candidate RNAs for U3 snoRNA from the immunoprecipitated RNAs of C. elegans using an antibody against the 2,2,7-trimethylguanosine (TMG) cap. The sequences were identical to the predicted U3 sequences in the RNomics study. Here, we show the several lines of evidence that the isolated RNAs are the true U3 snoRNAs of C. elegans. Moreover, we report the novel expression pattern of U3 snoRNA and fibrillarin, which is an essential component of U3 small nucleolar ribonucleoprotein complex, during early embryo development of C. elegans. To our knowledge, this is the first observation of the inconsistent localization U3 snoRNA and fibrillarin during early embryogenesis, providing novel insight into the mechanisms of nucleologenesis and ribosome production during early embryogenesis.

Combined in silico and experimental identification of the Pyrococcus abyssi H/ACA sRNAs and their target sites in ribosomal RNAs.

How far do H/ACA sRNPs contribute to rRNA pseudouridylation in Archaea was still an open question. Hence here, by computational search in three Pyrococcus genomes, we identified seven H/ACA sRNAs and predicted their target sites in rRNAs. In parallel, we experimentally identified 17 Psi residues in P. abyssi rRNAs. By in vitro reconstitution of H/ACA sRNPs, we assigned 15 out of the 17 Psi residues to the 7 identified H/ACA sRNAs: one H/ACA motif can guide up to three distinct pseudouridylations. Interestingly, by using a 23S rRNA fragment as the substrate, one of the two remaining Psi residues could be formed in vitro by the aCBF5/aNOP10/aGAR1 complex without guide sRNA. Our results shed light on structural constraints in archaeal H/ACA sRNPs: the length of helix H2 is of 5 or 6 bps, the distance between the ANA motif and the targeted U residue is of 14 or 15 nts, and the stability of the interaction formed by the substrate rRNA and the 3'-guide sequence is more important than that formed with the 5'-guide sequence. Surprisingly, we showed that a sRNA-rRNA interaction with the targeted uridine in a single-stranded 5'-UNN-3' trinucleotide instead of the canonical 5'-UN-3' dinucleotide is functional.