scaRNAs Research Articles

Non-coding RNA profiling of the developing murine lens

Non-coding RNAs (ncRNAs) are emerging as an important player in the regulation of genome integrity and gene expression, and they have been implicated in the pathogenesis of many diseases. The aim of the present study is to identify the repertoire of ncRNAs expressed in the developing mouse lens. We previously reported the mouse lens transcriptome, including mRNA and microRNA (miRNA) profiling at two embryonic (E15 and E18) and four postnatal (P0, P3, P6, and P9) time points. We analyzed the data from small RNA-Seq and mRNA-Seq libraries to investigate the ncRNA profile. Our analysis revealed expression of 12 different classes of ncRNA in the murine lens at six developmental time points. Annotation of small RNA data showed expression of 1,756 antisense ncRNA (asncRNA) in the mouse lens transcriptome. Likewise, we identified 82 P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA), 345 transfer RNA (tRNA), 12 small nuclear RNA (snRNA), 167 small nucleolar RNA (snoRNA), 19 small Cajal body-specific RNA (scaRNA), six ribosomal RNA (rRNA), 18 tRNA-like structures, one MALAT1-associated small cytoplasmic RNA (mascRNA), one Vault RNA (vtRNA), and one Y RNA expressed in the developing mouse lens. In parallel, bioinformatic investigation of mRNA-Seq data identified expression of 1,952 long intergenic ncRNA (lincRNA) in the developing mouse lens. In conclusion, we report a comprehensive ncRNA profile in the murine lens at six developmental time points. To the best of our knowledge, this is first report investigating different classes of ncRNAs in the developing mouse lens and will be monumental in elucidating processes essential for the development of the ocular lens and the maintenance of its transparency.

Promoter-based identification of novel non-coding RNAs reveals the presence of dicistronic snoRNA-miRNA genes in Arabidopsis thaliana.

BackgroundIn the past few decades, non-coding RNAs (ncRNAs) have emerged as important regulators of gene expression in eukaryotes. Most studies of ncRNAs in plants have focused on the identification of silencing microRNAs (miRNAs) and small interfering RNAs (siRNAs). Another important family of ncRNAs that has been well characterized in plants is the small nucleolar RNAs (snoRNAs) and the related small Cajal body-specific RNAs (scaRNAs). Both target chemical modifications of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). In plants, the snoRNA genes are organized in clusters, transcribed by RNA Pol II from a common promoter and subsequently processed into mature molecules. The promoter regions of snoRNA polycistronic genes in plants are highly enriched in two conserved cis-regulatory elements (CREs), Telo-box and Site II, which coordinate the expression of snoRNAs and ribosomal protein coding genes throughout the cell cycle.ResultsIn order to identify novel ncRNA genes, we have used the snoRNA Telo-box/Site II motifs combination as a functional promoter indicator to screen the Arabidopsis genome. The predictions generated by this process were tested by detailed exploration of available RNA-Seq and expression data sets and experimental validation. As a result, we have identified several snoRNAs, scaRNAs and 'orphan' snoRNAs. We also show evidence for 16 novel ncRNAs that lack similarity to any reported RNA family. Finally, we have identified two dicistronic genes encoding precursors that are processed to mature snoRNA and miRNA molecules. We discuss the evolutionary consequences of this result in the context of a tight link between snoRNAs and miRNAs in eukaryotes.ConclusionsWe present an alternative computational approach for non-coding RNA detection. Instead of depending on sequence or structure similarity in the whole genome screenings, we have explored the properties of promoter regions of well-characterized ncRNAs. Interestingly, besides expected ncRNAs predictions we were also able to recover single precursor arrangement for snoRNA-miRNA. Accompanied by analyses performed on rice sequences, we conclude that such arrangement might have interesting functional and evolutionary consequences and discuss this result in the context of a tight link between snoRNAs and miRNAs in eukaryotes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2221-x) contains supplementary material, which is available to authorized users.

ScaRNAs regulate splicing and vertebrate heart development

Alternative splicing (AS) plays an important role in regulating mammalian heart development, but a link between misregulated splicing and congenital heart defects (CHDs) has not been shown. We reported that more than 50% of genes associated with heart development were alternatively spliced in the right ventricle (RV) of infants with tetralogy of Fallot (TOF). Moreover, there was a significant decrease in the level of 12 small cajal body-specific RNAs (scaRNAs) that direct the biochemical modification of specific nucleotides in spliceosomal RNAs. We sought to determine if scaRNA levels influence patterns of AS and heart development. We used primary cells derived from the RV of infants with TOF to show a direct link between scaRNA levels and splice isoforms of several genes that regulate heart development (e.g., GATA4, NOTCH2, DAAM1, DICER1, MBNL1 and MBNL2). In addition, we used antisense morpholinos to knock down the expression of two scaRNAs (scarna1 and snord94) in zebrafish and saw a corresponding disruption of heart development with an accompanying alteration in splice isoforms of cardiac regulatory genes. Based on these combined results, we hypothesize that scaRNA modification of spliceosomal RNAs assists in fine tuning the spliceosome for dynamic selection of mRNA splice isoforms. Our results are consistent with disruption of splicing patterns during early embryonic development leading to insufficient communication between the first and second heart fields, resulting in conotruncal misalignment and TOF. Our findings represent a new paradigm for determining the mechanisms underlying congenital cardiac malformations.

Proteostatic Control of Telomerase Function through TRiC-Mediated Folding of TCAB1

Telomere maintenance by telomerase is impaired in the stem cell disease dyskeratosis congenita and during human aging. Telomerase depends upon a complex pathway for enzyme assembly, localization in Cajal bodies, and association with telomeres. Here, we identify the chaperonin CCT/TRiC as a critical regulator of telomerase trafficking using a high-content genome-wide siRNA screen in human cells for factors required for Cajal body localization. We find that TRiC is required for folding the telomerase cofactor TCAB1, which controls trafficking of telomerase and small Cajal body RNAs (scaRNAs). Depletion of TRiC causes loss of TCAB1 protein, mislocalization of telomerase and scaRNAs to nucleoli, and failure of telomere elongation. DC patient-derived mutations in TCAB1 impair folding by TRiC, disrupting telomerase function and leading to severe disease. Our findings establish a critical role for TRiC-mediated protein folding in the telomerase pathway and link proteostasis, telomere maintenance, and human disease.

Anesthesia for Euthanasia Influences mRNA Expression in Healthy Mice and after Traumatic Brain Injury

Tissue sampling for gene expression analysis is usually performed under general anesthesia. Anesthetics are known to modulate hemodynamics, receptor-mediated signaling cascades, and outcome parameters. The present study determined the influence of anesthetic paradigms typically used for euthanization and tissue sampling on cerebral mRNA expression in mice. Naïve mice and animals with acute traumatic brain injury induced by controlled cortical impact (CCI) were randomized to the following euthanasia protocols (n=10-11/group): no anesthesia (NA), 1 min of 4 vol% isoflurane in room air (ISO), 3 min of a combination of 5 mg/kg midazolam, 0.05 mg/kg fentanyl, and 0.5 mg/kg medetomidine intraperitoneally (COMB), or 3 min of 360 mg/kg chloral hydrate intraperitoneally (CH). mRNA expression of actin-1-related gene (Act1), FBJ murine osteosarcoma viral oncogene homolog B (FosB), tumor necrosis factor alpha (TNFα), heat shock protein beta-1 (HspB1), interleukin (IL)-6, tight junction protein 1 (ZO-1), IL-1ß, cyclophilin A, micro RNA 497 (miR497), and small cajal body-specific RNA 17 were determined by real-time polymerase chain reaction (PCR) in hippocampus samples. In naïve animals, Act1 expression was downregulated in the CH group compared with NA. FosB expression was downregulated in COMB and CH groups compared with NA. CCI reduced Act1 and FosB expression, whereas HspB1 and TNFα expression increased. After CCI, HspB1 expression was significantly higher in ISO, COMB, and CH groups, and TNFα expression was elevated in ISO and COMB groups. MiR497, IL-6, and IL-1ß were upregulated after CCI but not affected by anesthetics. Effects were independent of absolute mRNA copy numbers. The data demonstrate that a few minutes of anesthesia before tissue sampling are sufficient to induce immediate mRNA changes, which seem to predominate in the early-regulated gene cluster. Anesthesia-related effects on gene expression might explain limited reproduciblity of real-time PCR data between studies or research groups and should therefore be considered for quantitative PCR data.

Coilin association with Box C/D scaRNA suggests a direct role for the Cajal body marker protein in scaRNP biogenesis

ABSTRACTSpliceosomal small nuclear ribonucleoproteins (snRNPs) are enriched in the Cajal body (CB). Guide RNAs, known as small Cajal body-specific RNAs (scaRNAs), direct modification of the small nuclear RNA (snRNA) component of the snRNP. The protein WRAP53 binds a sequence motif (the CAB box) found in many scaRNAs and the RNA component of telomerase (hTR) and targets these RNAs to the CB. We have previously reported that coilin, the CB marker protein, associates with certain non-coding RNAs. For a more comprehensive examination of the RNAs associated with coilin, we have sequenced the RNA isolated from coilin immunocomplexes. A striking preferential association of coilin with the box C/D scaRNAs 2 and 9, which lack a CAB box, was observed. This association varied by treatment condition and WRAP53 knockdown. In contrast, reduction of WRAP53 did not alter the level of coilin association with hTR. Additional studies showed that coilin degrades/processes scaRNA 2 and 9, associates with active telomerase and can influence telomerase activity. These findings suggest that coilin plays a novel role in the biogenesis of box C/D scaRNPs and telomerase.

RIP-seq analysis of eukaryotic Sm proteins identifies three major categories of Sm-containing ribonucleoproteins.

BackgroundSm proteins are multimeric RNA-binding factors, found in all three domains of life. Eukaryotic Sm proteins, together with their associated RNAs, form small ribonucleoprotein (RNP) complexes important in multiple aspects of gene regulation. Comprehensive knowledge of the RNA components of Sm RNPs is critical for understanding their functions.ResultsWe developed a multi-targeting RNA-immunoprecipitation sequencing (RIP-seq) strategy to reliably identify Sm-associated RNAs from Drosophila ovaries and cultured human cells. Using this method, we discovered three major categories of Sm-associated transcripts: small nuclear (sn)RNAs, small Cajal body (sca)RNAs and mRNAs. Additional RIP-PCR analysis showed both ubiquitous and tissue-specific interactions. We provide evidence that the mRNA-Sm interactions are mediated by snRNPs, and that one of the mechanisms of interaction is via base pairing. Moreover, the Sm-associated mRNAs are mature, indicating a splicing-independent function for Sm RNPs.ConclusionsThis study represents the first comprehensive analysis of eukaryotic Sm-containing RNPs, and provides a basis for additional functional analyses of Sm proteins and their associated snRNPs outside of the context of pre-mRNA splicing. Our findings expand the repertoire of eukaryotic Sm-containing RNPs and suggest new functions for snRNPs in mRNA metabolism.

Novel small Cajal-body-specific RNAs identified in Drosophila: probing guide RNA function.

The spliceosomal small nuclear RNAs (snRNAs) are modified post-transcriptionally by introduction of pseudouridines and 2'-O-methyl modifications, which are mediated by box H/ACA and box C/D guide RNAs, respectively. Because of their concentration in the nuclear Cajal body (CB), these guide RNAs are known as small CB-specific (sca) RNAs. In the cell, scaRNAs are associated with the WD-repeat protein WDR79. We used coimmunoprecipitation with WDR79 to recover seven new scaRNAs from Drosophila cell lysates. We demonstrated concentration of these new scaRNAs in the CB by in situ hybridization, and we verified experimentally that they can modify their putative target RNAs. Surprisingly, one of the new scaRNAs targets U6 snRNA, whose modification is generally assumed to occur in the nucleolus, not in the CB. Two other scaRNAs have dual guide functions, one for an snRNA and one for 28S rRNA. Again, the modification of 28S rRNA is assumed to take place in the nucleolus. These findings suggest that canonical scaRNAs may have functions in addition to their established role in modifying U1, U2, U4, and U5 snRNAs. We discuss the likelihood that processing by scaRNAs is not limited to the CB.

Small nucleolar RNAs as new biomarkers in chronic lymphocytic leukemia.

BackgroundSmall nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs are non-coding RNAs involved in the maturation of other RNA molecules. Alterations of sno/scaRNA expression may play a role in cancerogenesis. This study elucidates the patterns of sno/scaRNA expression in 211 chronic lymphocytic leukemia (CLL) patients (Binet stage A) also in comparison with those of different normal B-cell subsets.MethodsThe patterns of sno/scaRNA expression in highly purified CD19+ B-cells of 211 CLL patients and in 18 normal B-cell samples - 6 from peripheral blood, and 12 from tonsils (4 germinal center, 2 marginal zone, 3 switched memory and 3 naïve B-cells) - were analyzed on the Affymetrix GeneChip® Human Gene 1.0 ST array.ResultsCLLs display a sno/scaRNAs expression profile similar to normal memory, naïve and marginal-zone B-cells, with the exception of a few down-regulated transcripts (SNORA31, -6, -62, and -71C). Our analyses also suggest some heterogeneity in the pattern of sno/scaRNAs expression which is apparently unrelated to the major biological (ZAP-70 and CD38), molecular (IGHV mutation) and cytogenetic markers. Moreover, we found that SNORA70F was significantly down-regulated in poor prognostic subgroups and this phenomenon was associated with the down-regulation of its host gene COBLL1. Finally, we generated an independent model based on SNORA74A and SNORD116-18 expression, which appears to distinguish two different prognostic CLL groups.ConclusionsThese data extend the view of sno/scaRNAs deregulation in cancer and may contribute to discover novel biomarkers associated with the disease and potentially useful to predict the clinical outcome of early stage CLL patients.

The Expression Pattern of Small Nucleolar and Small Cajal Body-Specific RNAs Characterizes Distinct Molecular Subtypes of Multiple Myeloma

AbstractAbstract 3955Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs involved in the maturation of other RNA molecules and generally located in the introns of host genes. It is an emerging evidence that altered sno/scaRNAs expression may play a pathological role in cancer. Impaired sno/scaRNAs expression has recently been reported both in acute leukemia and smoldering myeloma that rapidly progressed to symptomatic disease. In addition, as regards multiple myeloma (MM), very recent data suggested an oncogenic role for SCARNA22 in those MM patients over-expressing SCARNA22/MMSET as a result of t(4;14) translocation. However, comprehensive information concerning the expression behavior of sno/scaRNAs in MM is still lacking.This study elucidates the patterns of sno/scaRNAs expression in MM by profiling purified malignant plasma cells from 55 MMs, 8 secondary plasma cell leukemias (sPCL) and 4 normal controls using Human Gene 1.0 ST arrays.Overall, a global sno/scaRNAs down-regulation was found in MMs and at more extent in sPCLs compared to normal plasma cells. Whereas SCARNA22 resulted the only sno/scaRNA characterizing the TC4 MM, TC2 group displayed a distinct sno/scaRNA signature overexpressing members of SNORD115 and SNORD116 families located in a region finely regulated by an imprinting center at 15q11 which, however, resulted overall hypomethylated in MMs independently of the SNORD115 and SNORD116 expression levels. In addition, impaired expression of sno/scaRNAs raised from the comparison between MM and sPCL, suggested a role in tumor progression. Furthermore, to uncover possible mechanisms at the basis of sno/scaRNAs deregulation, we investigated the correlation between sno/scaRNAs and the corresponding host-genes expression levels, outlining the coordinated expression of up to 50% of sno/scaRNAs/host-genes pairs. Finally, we investigated whether the sno/scaRNAs transcriptional pattern may be influenced by allelic imbalances involving their genomic location, as already demonstrated concerning mRNA expression, and revealed a dosage effect involving several chromosomal regions.Our data extend the current view of sno/scaRNAs deregulation in cancer and add novel information into the bio-molecular complexity of plasma cell dyscrasias. Furthermore, our findings may contribute to develop functional approaches to examine the activity of deregulated sno/scaRNAs in MM, as well as to further enlighten their possible role as targets of novel therapeutic agents. Disclosures:No relevant conflicts of interest to declare.

Pearls are novel Cajal body-like structures in the Xenopus germinal vesicle that are dependent on RNA pol III transcription

We have identified novel nuclear bodies, which we call pearls, in the giant oocyte nuclei of Xenopus laevis and Xenopus tropicalis. Pearls are attached to the lampbrush chromosomes at specific loci that are transcribed by RNA polymerase III, and they disappear after inhibition of polymerase III activity. Pearls are enriched for small Cajal body-specific RNAs (scaRNAs), which are guide RNAs that modify specific nucleotides on splicing snRNAs. Surprisingly, snRNAs themselves are not present in pearls, suggesting that pearls are not functionally equivalent to Cajal bodies in other systems, which contain both snRNAs and scaRNAs. We suggest that pearls may function in the processing of RNA polymerase III transcripts, such as tRNA, 5S rRNA, and other short non-coding RNAs.

The expression pattern of small nucleolar and small Cajal body-specific RNAs characterizes distinct molecular subtypes of multiple myeloma

Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs involved in the maturation of other RNA molecules and generally located in the introns of host genes. It is now emerging that altered sno/scaRNAs expression may have a pathological role in cancer. This study elucidates the patterns of sno/scaRNAs expression in multiple myeloma (MM) by profiling purified malignant plasma cells from 55 MMs, 8 secondary plasma cell leukemias (sPCLs) and 4 normal controls. Overall, a global sno/scaRNAs downregulation was found in MMs and, even more, in sPCLs compared with normal plasma cells. Whereas SCARNA22 resulted the only sno/scaRNA characterizing the translocation/cyclin D4 (TC4) MM, TC2 group displayed a distinct sno/scaRNA signature overexpressing members of SNORD115 and SNORD116 families located in a region finely regulated by an imprinting center at 15q11, which, however, resulted overall hypomethylated in MMs independently of the SNORD115 and SNORD116 expression levels. Finally, integrative analyses with available gene expression and genome-wide data revealed the occurrence of significant sno/scaRNAs/host genes co-expression and the putative influence of allelic imbalances on specific snoRNAs expression. Our data extend the current view of sno/scaRNAs deregulation in cancer and add novel information to the bio-molecular complexity of plasma cell dyscrasias.

Maturation of mammalian H/ACA box snoRNAs: PAPD5-dependent adenylation and PARN-dependent trimming

Small nucleolar and small Cajal body RNAs (snoRNAs and scaRNAs) of the H/ACA box and C/D box type are generated by exonucleolytic shortening of longer precursors. Removal of the last few nucleotides at the 3' end is known to be a distinct step. We report that, in human cells, knock-down of the poly(A) specific ribonuclease (PARN), previously implicated only in mRNA metabolism, causes the accumulation of oligoadenylated processing intermediates of H/ACA box but not C/D box RNAs. In agreement with a role of PARN in snoRNA and scaRNA processing, the enzyme is concentrated in nucleoli and Cajal bodies. Oligo(A) tails are attached to a short stub of intron sequence remaining beyond the mature 3' end of the snoRNAs. The noncanonical poly(A) polymerase PAPD5 is responsible for addition of the oligo(A) tails. We suggest that deadenylation is coupled to clean 3' end trimming, which might serve to enhance snoRNA stability.

Post-transcriptional modification of spliceosomal RNAs is normal in SMN-deficient cells

The survival of motor neuron (SMN) protein plays an important role in the biogenesis of spliceosomal snRNPs and is one factor required for the integrity of nuclear Cajal bodies (CBs). CBs are enriched in small CB-specific (sca) RNAs, which guide the formation of pseudouridylated and 2'-O-methylated residues in the snRNAs. Because SMN-deficient cells lack typical CBs, we asked whether the modification of internal residues of major and minor snRNAs is defective in these cells. We mapped modified nucleotides in the major U2 and the minor U4atac and U12 snRNAs. Using both radioactive and fluorescent primer extension approaches, we found that modification of major and minor spliceosomal snRNAs is normal in SMN-deficient cells. Our experiments also revealed a previously undetected pseudouridine at position 60 in human U2 and 2'-O-methylation of A1, A2, and G19 in human U4atac. These results confirm, and extend to minor snRNAs, previous experiments showing that scaRNPs can function in the absence of typical CBs. Furthermore, they show that the differential splicing defects in SMN-deficient cells are not due to failure of post-transcriptional modification of either major or minor snRNAs.

Architecture of human telomerase RNA

Telomerase is a unique reverse transcriptase that catalyzes the addition of telomere DNA repeats onto the 3' ends of linear chromosomes and plays a critical role in maintaining genome stability. Unlike other reverse transcriptases, telomerase is unique in that it is a ribonucleoprotein complex, where the RNA component [telomerase RNA (TR)] not only provides the template for the synthesis of telomere DNA repeats but also plays essential roles in catalysis, accumulation, TR 3'-end processing, localization, and holoenzyme assembly. Biochemical studies have identified TR elements essential for catalysis that share remarkably conserved secondary structures across different species as well as species-specific domains for other functions, paving the way for high-resolution structure determination of TRs. Over the past decade, structures of key elements from the core, conserved regions 4 and 5, and small Cajal body specific RNA domains of human TR have emerged, providing significant insights into the roles of these RNA elements in telomerase function. Structures of all helical elements of the core domain have been recently reported, providing the basis for a high-resolution model of the complete core domain. We review this progress to determine the overall architecture of human telomerase RNA.

WRAP53 promotes cancer cell survival and is a potential target for cancer therapy

We previously identified WRAP53 as an antisense transcript that regulates the p53 tumor suppressor. The WRAP53 gene also encodes a protein essential for Cajal body formation and involved in cellular trafficking of the survival of motor neuron complex, the telomerase enzyme and small Cajal body-specific RNAs to Cajal bodies. Here, we show that the WRAP53 protein is overexpressed in a variety of cancer cell lines of different origin and that WRAP53 overexpression promotes cellular transformation. Knockdown of the WRAP53 protein triggers massive apoptosis through the mitochondrial pathway, as demonstrated by Bax/Bak activation, loss of mitochondrial membrane potential and cytochrome c release. The apoptosis induced by WRAP53 knockdown could moreover be blocked by Bcl-2 overexpression. Interestingly, human tumor cells are more sensitive to WRAP53 depletion as compared with normal human cells indicating that cancer cells in particular depends on WRAP53 expression for their survival. In agreement with this, we found that high levels of WRAP53 correlate with poor prognosis of head and neck cancer. Together these observations propose a role of WRAP53 in carcinogenesis and identify WRAP53 as a novel molecular target for a large fraction of malignancies.

Mutations In TCAB1 Cause Dyskeratosis Congenita

AbstractAbstract 197Dyskeratosis congenita (DC) is a multisystem disorder characterized by the diagnostic triad of nail dysplasia, abnormal skin pigmentation, and oral leukoplakia. Patients with DC are at very high risk of bone marrow failure, cancer, pulmonary fibrosis, and other complications. All patients with DC have very short telomeres; approximately 60% have a mutation in one of 6 telomere biology genes (DCK1, TERC, TERT, TINF2, NOP10 or NHP2). Telomerase (TERT) is the reverse transcriptase which, with its RNA component TERC, extends telomeric repeats to offset the telomere shortening that occurs with DNA replication. Mutations in the catalytic core component of TERT (autosomal dominant or recessive) or TERC (autosomal dominant) cause DC and related telomere biology disorders. The function of the telomerase enzyme in vivo requires additional components including dyskerin (DKC1), which is mutated in patients with X-linked recessive DC. Dyskerin binds the H/ACA sequence within TERC and is required for the biogenesis of telomerase, as well as the biogenesis and function of other RNP complexes, including small nucleolar RNAs (snoRNAs) and small Cajal body RNAs (scaRNAs), involved in the modification of ribosomal RNAs and splicing RNAs, respectively. We recently identified TCAB1 (gene names WDR79, WRAP53) as a novel telomerase component through biochemical purification of telomerase complexes. TCAB1 is a WD40-repeat containing protein that binds the CAB box sequence within TERC. It is a constituent of the active telomerase holoenzyme and inhibition of TCAB1 prevents telomerase from localizing to Cajal bodies where RNA-protein complexes are assembled and modified.Since TCAB1 is required for telomerase trafficking, we evaluated mutations in TCAB1 as a potential cause of DC in 16 mutation-negative patients who were participants in the NCI's prospective Inherited Bone Marrow Failure Syndromes (IBMFS) study. Nine patients had classic DC, defined by the diagnostic triad, or the combination of 1 of the triad plus BMF and 2 other DC-related complications, and leukocyte telomeres <1st percentile for age. An additional 7 DC-like patients (lacking the diagnostic triad) with BMF and telomeres <1st percentile were also evaluated. We performed bi-directional sequence analysis of all 10 exons of TCAB1; PCR and sequence analysis were independently repeated 3 times and reviewed by individuals blinded to patient status. Two unrelated patients with classic DC and very short telomeres had different compound heterozygous mutations in TCAB1. Their parents were not consanguineous and had normal telomere lengths. Each healthy parent harbored a single heterozygous mutation in TCAB1 consistent with autosomal recessive inheritance. These mutations were not present in 380 healthy controls. Each of the four disease-associated TCAB1 alleles showed a defect in accumulation and localization within the Cajal body. Mutant TCAB1 alleles were stably expressed in HeLa cells, but showed markedly reduced accumulation in Cajal bodies by immunofluorescence compared with wild-type TCAB1. Analysis of endogenous TCAB1 expression in TCAB1-mutant, EBV-immortalized lymphoblasts similarly showed a marked impairment of TCAB1 accumulation in Cajal bodies. To model the effects of impaired TCAB1 function, we depleted TCAB1 using siRNA in HeLa cells. TCAB1 depletion prevented TERC from localizing in Cajal bodies, and instead caused TERC to accumulate in nucleoli, as judged by RNA fluorescence in situ hybridization for TERC RNA. Analysis of TERC localization in TCAB1-mutant DC patient lymphoblasts similarly showed that TERC aberrantly accumulated in nucleoli, rather than in Cajal bodies (p<0.001). The overall levels of TERT RNA were unaltered in TCAB1-mutant lymphoblasts by Northern blot. Our data show that compound heterozygosity for TCAB1 mutations leads to a marked mislocalization of telomerase and can cause classic DC. Our findings identify telomerase mislocalization as a novel cause of disease and provide a framework for investigating telomerase trafficking in DC and other disease states. Disclosures:No relevant conflicts of interest to declare.

WRAP53 Is Essential for Cajal Body Formation and for Targeting the Survival of Motor Neuron Complex to Cajal Bodies

Author SummaryCajal bodies, discovered more than 100 years ago by Santiago Ramón y Cajal, are sub-organelles found in the nucleus of proliferative cells and neurons. They have been implicated in a variety of nuclear functions including ribonucleoprotein maturation, spliceosome formation, histone mRNA processing, RNA polymerase assembly, telomerase biogenesis, and histone gene transcription. Concentrating relevant molecules within Cajal bodies may serve to increase the efficiency of specific nuclear functions. Here we identify the WRAP53 protein as an essential factor for Cajal body maintenance and for directing the splicing regulatory protein “survival of motor neuron” (SMN) complex to Cajal bodies. We show that WRAP53 is a constitutive component of Cajal bodies, and that knockdown of WRAP53 disrupts existing Cajal bodies and prevents formation of new Cajal bodies. Mechanistically, we find that WRAP53 recruits the SMN complex from the cytoplasm to Cajal bodies by mediating interactions between SMN, importinβ, and coilin. Finally, we report deficient WRAP53–SMN binding in patients with spinal muscular atrophy, suggesting a role in this pathology. This study not only reveals new functions of the WRAP53 protein, but also increases our understanding of the molecular mechanism behind Cajal body formation and recruitment of factors to Cajal bodies.

Plant U13 orthologues and orphan snoRNAs identified by RNomics of RNA from Arabidopsis nucleoli.

Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs whose main function in eukaryotes is to guide the modification of nucleotides in ribosomal and spliceosomal small nuclear RNAs, respectively. Full-length sequences of Arabidopsis snoRNAs and scaRNAs have been obtained from cDNA libraries of capped and uncapped small RNAs using RNA from isolated nucleoli from Arabidopsis cell cultures. We have identified 31 novel snoRNA genes (9 box C/D and 22 box H/ACA) and 15 new variants of previously described snoRNAs. Three related capped snoRNAs with a distinct gene organization and structure were identified as orthologues of animal U13snoRNAs. In addition, eight of the novel genes had no complementarity to rRNAs or snRNAs and are therefore putative orphan snoRNAs potentially reflecting wider functions for these RNAs. The nucleolar localization of a number of the snoRNAs and the localization to nuclear bodies of two putative scaRNAs was confirmed by in situ hybridization. The majority of the novel snoRNA genes were found in new gene clusters or as part of previously described clusters. These results expand the repertoire of Arabidopsis snoRNAs to 188 snoRNA genes with 294 gene variants.

A global view of the nonprotein-coding transcriptome in Plasmodium falciparum

Nonprotein-coding RNAs (npcRNAs) represent an important class of regulatory molecules that act in many cellular pathways. Here, we describe the experimental identification and validation of the small npcRNA transcriptome of the human malaria parasite Plasmodium falciparum. We identified 630 novel npcRNA candidates. Based on sequence and structural motifs, 43 of them belong to the C/D and H/ACA-box subclasses of small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs). We further observed the exonization of a functional H/ACA snoRNA gene, which might contribute to the regulation of ribosomal protein L7a gene expression. Some of the small npcRNA candidates are from telomeric and subtelomeric repetitive regions, suggesting their potential involvement in maintaining telomeric integrity and subtelomeric gene silencing. We also detected 328 cis-encoded antisense npcRNAs (asRNAs) complementary to P. falciparum protein-coding genes of a wide range of biochemical pathways, including determinants of virulence and pathology. All cis-encoded asRNA genes tested exhibit lifecycle-specific expression profiles. For all but one of the respective sense–antisense pairs, we deduced concordant patterns of expression. Our findings have important implications for a better understanding of gene regulatory mechanisms in P. falciparum, revealing an extended and sophisticated npcRNA network that may control the expression of housekeeping genes and virulence factors.