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• W2156880688 abstract "Thousands of long intervening noncoding RNAs (lincRNAs) have been identified in mammals. To better understand the evolution and functions of these enigmatic RNAs, we used chromatin marks, poly(A)-site mapping and RNA-Seq data to identify more than 550 distinct lincRNAs in zebrafish. Although these shared many characteristics with mammalian lincRNAs, only 29 had detectable sequence similarity with putative mammalian orthologs, typically restricted to a single short region of high conservation. Other lincRNAs had conserved genomic locations without detectable sequence conservation. Antisense reagents targeting conserved regions of two zebrafish lincRNAs caused developmental defects. Reagents targeting splice sites caused the same defects and were rescued by adding either the mature lincRNA or its human or mouse ortholog. Our study provides a roadmap for identification and analysis of lincRNAs in model organisms and shows that lincRNAs play crucial biological roles during embryonic development with functionality conserved despite limited sequence conservation. Thousands of long intervening noncoding RNAs (lincRNAs) have been identified in mammals. To better understand the evolution and functions of these enigmatic RNAs, we used chromatin marks, poly(A)-site mapping and RNA-Seq data to identify more than 550 distinct lincRNAs in zebrafish. Although these shared many characteristics with mammalian lincRNAs, only 29 had detectable sequence similarity with putative mammalian orthologs, typically restricted to a single short region of high conservation. Other lincRNAs had conserved genomic locations without detectable sequence conservation. Antisense reagents targeting conserved regions of two zebrafish lincRNAs caused developmental defects. Reagents targeting splice sites caused the same defects and were rescued by adding either the mature lincRNA or its human or mouse ortholog. Our study provides a roadmap for identification and analysis of lincRNAs in model organisms and shows that lincRNAs play crucial biological roles during embryonic development with functionality conserved despite limited sequence conservation. The zebrafish genome encodes hundreds of long intervening noncoding RNAs (lincRNAs) Only 29 of 567 lincRNAs have detectable sequence homology with mammalian lincRNAs Two lincRNAs, cyrano and megamind, are required for proper embryonic development The functionality of these two lincRNAs is retained in their human/mouse orthologs The availability of sequenced genomes for many species has shifted the focus from determining the genetic makeup of organisms to the delineation of the functional elements they encode. These analyses have revealed that, in addition to loci generating known genes, many other loci are transcribed, often in a regulated and tissue-specific fashion (Bertone et al., 2004Bertone P. Stolc V. Royce T.E. Rozowsky J.S. Urban A.E. Zhu X. Rinn J.L. Tongprasit W. Samanta M. Weissman S. et al.Global identification of human transcribed sequences with genome tiling arrays.Science. 2004; 306: 2242-2246Crossref PubMed Scopus (870) Google Scholar, Carninci et al., 2005Carninci P. Kasukawa T. Katayama S. Gough J. Frith M.C. Maeda N. Oyama R. Ravasi T. Lenhard B. Wells C. et al.The transcriptional landscape of the mammalian genome.Science. 2005; 309: 1559-1563Crossref PubMed Scopus (2794) Google Scholar, Dinger et al., 2008Dinger M.E. Amaral P.P. Mercer T.R. Pang K.C. Bruce S.J. Gardiner B.B. Askarian-Amiri M.E. Ru K. Solda G. Simons C. et al.Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation.Genome Res. 2008; 18: 1433-1445Crossref PubMed Scopus (641) Google Scholar, Mercer et al., 2008Mercer T.R. Dinger M.E. Sunkin S.M. Mehler M.F. Mattick J.S. Specific expression of long noncoding RNAs in the mouse brain.Proc. Natl. Acad. Sci. USA. 2008; 105: 716-721Crossref PubMed Scopus (934) Google Scholar, De Lucia and Dean, 2011De Lucia F. Dean C. Long non-coding RNAs and chromatin regulation.Curr. Opin. Plant Biol. 2011; 14: 168-173Crossref PubMed Scopus (71) Google Scholar, Jouannet and Crespi, 2011Jouannet V. Crespi M. Long nonprotein-coding RNAs in plants.Prog. Mol. Subcell. Biol. 2011; 51: 179-200Crossref PubMed Scopus (10) Google Scholar). In the human and mouse genomes, thousands of loci produce RNA molecules longer than 200 nucleotides (nt) that are capped, polyadenylated and often spliced, yet do not overlap protein-coding genes or previously characterized classes of noncoding RNAs (ncRNAs); these have been called long intervening ncRNAs (lincRNAs) (Guttman et al., 2009Guttman M. Amit I. Garber M. French C. Lin M.F. Feldser D. Huarte M. Zuk O. Carey B.W. Cassady J.P. et al.Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals.Nature. 2009; 458: 223-227Crossref PubMed Scopus (3277) Google Scholar, Khalil et al., 2009Khalil A.M. Guttman M. Huarte M. Garber M. Raj A. Rivea Morales D. Thomas K. Presser A. Bernstein B.E. van Oudenaarden A. et al.Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.Proc. Natl. Acad. Sci. USA. 2009; 106: 11667-11672Crossref PubMed Scopus (2359) Google Scholar). Although a few dozen mammalian lincRNAs have been characterized to some extent and reported to function in important cellular processes such as X chromosome inactivation, imprinting, pluripotency maintenance, and transcriptional regulation (Rinn et al., 2007Rinn J.L. Kertesz M. Wang J.K. Squazzo S.L. Xu X. Brugmann S.A. Goodnough L.H. Helms J.A. Farnham P.J. Segal E. et al.Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs.Cell. 2007; 129: 1311-1323Abstract Full Text Full Text PDF PubMed Scopus (3394) Google Scholar, Mercer et al., 2009Mercer T.R. Dinger M.E. Mattick J.S. Long non-coding RNAs: insights into functions.Nat. Rev. Genet. 2009; 10: 155-159Crossref PubMed Scopus (4447) Google Scholar, Gupta et al., 2010Gupta R.A. Shah N. Wang K.C. Kim J. Horlings H.M. Wong D.J. Tsai M.C. Hung T. Argani P. Rinn J.L. et al.Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis.Nature. 2010; 464: 1071-1076Crossref PubMed Scopus (4166) Google Scholar, Huarte et al., 2010Huarte M. Guttman M. Feldser D. Garber M. Koziol M.J. Kenzelmann-Broz D. Khalil A.M. Zuk O. Amit I. Rabani M. et al.A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response.Cell. 2010; 142: 409-419Abstract Full Text Full Text PDF PubMed Scopus (1703) Google Scholar, Orom et al., 2010Orom U.A. Derrien T. Beringer M. Gumireddy K. Gardini A. Bussotti G. Lai F. Zytnicki M. Notredame C. Huang Q. et al.Long noncoding RNAs with enhancer-like function in human cells.Cell. 2010; 143: 46-58Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar, Guttman et al., 2011Guttman M. Donaghey J. Carey B.W. Garber M. Grenier J.K. Munson G. Young G. Lucas A.B. Ach R. Bruhn L. et al.lincRNAs act in the circuitry controlling pluripotency and differentiation.Nature. 2011; 477: 295-300Crossref PubMed Scopus (1576) Google Scholar, Hung et al., 2011Hung T. Wang Y. Lin M.F. Koegel A.K. Kotake Y. Grant G.D. Horlings H.M. Shah N. Umbricht C. Wang P. et al.Extensive and coordinated transcription of noncoding RNAs within cell-cycle promoters.Nat. Genet. 2011; 43: 621-629Crossref PubMed Scopus (937) Google Scholar), the functions of most annotated lincRNAs are unknown. Comparative sequence analysis and functional studies in nonmammalian species have greatly advanced the understanding of protein-coding genes as well as microRNAs and other ncRNAs. However, these approaches were not immediately applied to lincRNAs because of their more limited sequence conservation (Ponjavic et al., 2007Ponjavic J. Ponting C.P. Lunter G. Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs.Genome Res. 2007; 17: 556-565Crossref PubMed Scopus (564) Google Scholar, Marques and Ponting, 2009Marques A.C. Ponting C.P. Catalogues of mammalian long noncoding RNAs: modest conservation and incompleteness.Genome Biol. 2009; 10: R124Crossref PubMed Scopus (208) Google Scholar). Thousands of lincRNAs have been reported in human and mouse, some of which have recognizable sequence homology in the other species (Ponjavic et al., 2007Ponjavic J. Ponting C.P. Lunter G. Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs.Genome Res. 2007; 17: 556-565Crossref PubMed Scopus (564) Google Scholar, Khalil et al., 2009Khalil A.M. Guttman M. Huarte M. Garber M. Raj A. Rivea Morales D. Thomas K. Presser A. Bernstein B.E. van Oudenaarden A. et al.Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.Proc. Natl. Acad. Sci. USA. 2009; 106: 11667-11672Crossref PubMed Scopus (2359) Google Scholar, Marques and Ponting, 2009Marques A.C. Ponting C.P. Catalogues of mammalian long noncoding RNAs: modest conservation and incompleteness.Genome Biol. 2009; 10: R124Crossref PubMed Scopus (208) Google Scholar, Guttman et al., 2010Guttman M. Garber M. Levin J.Z. Donaghey J. Robinson J. Adiconis X. Fan L. Koziol M.J. Gnirke A. Nusbaum C. et al.Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs.Nat. Biotechnol. 2010; 28: 503-510Crossref PubMed Scopus (982) Google Scholar). However, there have been only a few hints that orthologs of mammalian lincRNAs exist outside of mammals (Chodroff et al., 2010Chodroff R.A. Goodstadt L. Sirey T.M. Oliver P.L. Davies K.E. Green E.D. Molnar Z. Ponting C.P. Long noncoding RNA genes: conservation of sequence and brain expression among diverse amniotes.Genome Biol. 2010; 11: R72Crossref PubMed Scopus (194) Google Scholar, Stadler, 2010Stadler P.F. Evolution of the long non-coding RNAs MALAT1 and MENβ/ε.in: Ferreira C.E. Miyano S. Stadler P.F. Advances in Bioinformatics and Computational Biology. Springer, Rio de Janeiro, Brazil2010Google Scholar, Wang et al., 2011Wang K.C. Yang Y.W. Liu B. Sanyal A. Corces-Zimmerman R. Chen Y. Lajoie B.R. Protacio A. Flynn R.A. Gupta R.A. et al.A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression.Nature. 2011; 472: 120-124Crossref PubMed Scopus (1531) Google Scholar). Therefore, the promise of model organisms for providing insight into mammalian lincRNA genomics, evolution, and function has awaited accurate experimental identification of lincRNAs in a nonmammalian model organism. Although high-throughput RNA sequencing (RNA-Seq) provides information useful for lincRNA identification (Guttman et al., 2010Guttman M. Garber M. Levin J.Z. Donaghey J. Robinson J. Adiconis X. Fan L. Koziol M.J. Gnirke A. Nusbaum C. et al.Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs.Nat. Biotechnol. 2010; 28: 503-510Crossref PubMed Scopus (982) Google Scholar), the short reads of current technologies limit the ability to accurately delineate full-length transcriptional units, especially those of lincRNAs, which typically are expressed at low levels (Guttman et al., 2009Guttman M. Amit I. Garber M. French C. Lin M.F. Feldser D. Huarte M. Zuk O. Carey B.W. Cassady J.P. et al.Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals.Nature. 2009; 458: 223-227Crossref PubMed Scopus (3277) Google Scholar, Guttman et al., 2010Guttman, M., Garber, M., Levin, J.Z., Donaghey, J., Robinson, J., Adiconis, X., Fan, L., Koziol, M.J., Gnirke, A., Nusbaum, C., et al. (2010). Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nat. Biotechnol. 28, 503–510.Google Scholar, Khalil et al., 2009Khalil A.M. Guttman M. Huarte M. Garber M. Raj A. Rivea Morales D. Thomas K. Presser A. Bernstein B.E. van Oudenaarden A. et al.Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.Proc. Natl. Acad. Sci. USA. 2009; 106: 11667-11672Crossref PubMed Scopus (2359) Google Scholar). Therefore, to build a robust pipeline for lincRNA discovery, complementary datasets augmenting RNA-Seq–based reconstruction must be acquired and integrated. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) generates genome-wide chromatin-state maps (Barski et al., 2007Barski A. Cuddapah S. Cui K. Roh T.Y. Schones D.E. Wang Z. Wei G. Chepelev I. Zhao K. High-resolution profiling of histone methylations in the human genome.Cell. 2007; 129: 823-837Abstract Full Text Full Text PDF PubMed Scopus (5056) Google Scholar, Mikkelsen et al., 2007Mikkelsen T.S. Ku M. Jaffe D.B. Issac B. Lieberman E. Giannoukos G. Alvarez P. Brockman W. Kim T.K. Koche R.P. et al.Genome-wide maps of chromatin state in pluripotent and lineage-committed cells.Nature. 2007; 448: 553-560Crossref PubMed Scopus (3235) Google Scholar), which enabled the preliminary annotation of many mammalian lincRNAs (Guttman et al., 2009Guttman M. Amit I. Garber M. French C. Lin M.F. Feldser D. Huarte M. Zuk O. Carey B.W. Cassady J.P. et al.Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals.Nature. 2009; 458: 223-227Crossref PubMed Scopus (3277) Google Scholar, Khalil et al., 2009Khalil A.M. Guttman M. Huarte M. Garber M. Raj A. Rivea Morales D. Thomas K. Presser A. Bernstein B.E. van Oudenaarden A. et al.Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.Proc. Natl. Acad. Sci. USA. 2009; 106: 11667-11672Crossref PubMed Scopus (2359) Google Scholar). Particularly informative have been the maps of histone H3 lysine 4 trimethylation (H3K4me3), which marks promoters of genes actively transcribed by RNA polymerase II, and maps of histone H3 lysine 36 trimethylation (H3K36me3), which marks the bodies of these genes (Schubeler et al., 2004Schubeler D. MacAlpine D.M. Scalzo D. Wirbelauer C. Kooperberg C. van Leeuwen F. Gottschling D.E. O'Neill L.P. Turner B.M. Delrow J. et al.The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote.Genes Dev. 2004; 18: 1263-1271Crossref PubMed Scopus (637) Google Scholar, Marson et al., 2008Marson A. Levine S.S. Cole M.F. Frampton G.M. Brambrink T. Johnstone S. Guenther M.G. Johnston W.K. Wernig M. Newman J. et al.Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells.Cell. 2008; 134: 521-533Abstract Full Text Full Text PDF PubMed Scopus (1203) Google Scholar). Another approach that provides important information for defining boundaries of transcriptional units is poly(A)-position profiling by sequencing (3P-Seq) (Jan et al., 2011Jan C.H. Friedman R.C. Ruby J.G. Bartel D.P. Formation, regulation and evolution of Caenorhabditis elegans 3′UTRs.Nature. 2011; 469: 97-101Crossref PubMed Scopus (342) Google Scholar). This method isolates distal segments of polyadenylated transcripts and identifies them by high-throughput sequencing. Although the initial description and application of 3P-Seq focused on protein-coding genes (Jan et al., 2011Jan C.H. Friedman R.C. Ruby J.G. Bartel D.P. Formation, regulation and evolution of Caenorhabditis elegans 3′UTRs.Nature. 2011; 469: 97-101Crossref PubMed Scopus (342) Google Scholar), the method defines 3′ termini of all polyadenylated transcripts, including lincRNAs. To identify lincRNAs of zebrafish (Danio rerio), we acquired chromatin maps and poly(A) sites from three developmental stages and developed a framework for lincRNA discovery that integrates these new datasets with transcriptome datasets, which included RNA-Seq reads, annotated ESTs and full-length cDNAs. We report more than 550 distinct lincRNAs in zebrafish and analyze their sequence and genomic properties, temporal and spatial expression, and conservation. For functional studies, we examined two lincRNAs with short stretches of deep conservation across vertebrates and found that these lincRNAs play important roles in brain morphogenesis and eye development, and that these functions are retained in their mammalian orthologs. To allow a systematic overview of actively transcribed regions, we generated genome-wide chromatin maps of histone H3 modifications with ChIP-Seq, focusing on zebrafish embryos at 24 and 72 hr postfertilization (hpf) and mixed-gender adults (Table S1 available online). As in other species (Barski et al., 2007Barski A. Cuddapah S. Cui K. Roh T.Y. Schones D.E. Wang Z. Wei G. Chepelev I. Zhao K. High-resolution profiling of histone methylations in the human genome.Cell. 2007; 129: 823-837Abstract Full Text Full Text PDF PubMed Scopus (5056) Google Scholar, Marson et al., 2008Marson A. Levine S.S. Cole M.F. Frampton G.M. Brambrink T. Johnstone S. Guenther M.G. Johnston W.K. Wernig M. Newman J. et al.Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells.Cell. 2008; 134: 521-533Abstract Full Text Full Text PDF PubMed Scopus (1203) Google Scholar, Kolasinska-Zwierz et al., 2009Kolasinska-Zwierz P. Down T. Latorre I. Liu T. Liu X.S. Ahringer J. Differential chromatin marking of introns and expressed exons by H3K36me3.Nat. Genet. 2009; 41: 376-381Crossref PubMed Scopus (496) Google Scholar), H3K4me3 marks were strongly enriched around transcription start sites, H3K36me3 levels were higher in gene bodies, and the amplitudes of both marks reflected gene expression levels (Figures S1A and S1B ). At each stage, between 16,171 and 19,557 H3K4me3 peaks were identified (Figure 1A and Table S2), most of which were present in all three stages (Figure S1C). We also applied 3P-Seq to poly(A)-selected RNA from the same three stages and identified 66,895 poly(A) sites (Figure 1B and Table S1 and Table S2).Figure 1Identification of Zebrafish lincRNA GenesShow full caption(A) Positions of H3K4me3 peaks from 24-hpf embryos with respect to annotations of known protein-coding genes and genes of small ncRNAs (<200 nt) annotated in Ensembl or RefSeq.(B) Positions of poly(A) sites with respect to annotated protein-coding and small ncRNA genes.(C) Pipeline for identification of lincRNAs. See text and extended experimental procedures for description.See also Figure S1 and Table S2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Positions of H3K4me3 peaks from 24-hpf embryos with respect to annotations of known protein-coding genes and genes of small ncRNAs (<200 nt) annotated in Ensembl or RefSeq. (B) Positions of poly(A) sites with respect to annotated protein-coding and small ncRNA genes. (C) Pipeline for identification of lincRNAs. See text and extended experimental procedures for description. See also Figure S1 and Table S2. Although the majority of H3K4me3 peaks and poly(A) sites could be assigned to known genes, a significant fraction occurred in regions without transcript annotation (Figures 1A and 1B). Poly(A) sites that could not be assigned to protein-coding or microRNA genes were used as seeds for identification of lincRNAs (Figure 1C), because sites identified by 3P-Seq unambiguously determine the strand of the transcript and one of its termini, which substantially constrained the subsequent search space. For each of these poly(A) sites, the closest upstream H3K4me3 peaks were identified, and putative lincRNA domains were defined as regions spanning from an H3K4me3 peak to a poly(A) site. After filtering out domains overlapping exons of protein-coding genes or small RNAs in the sense orientation, or coding exons in the antisense orientation, the remaining domains were significantly enriched for H3K36me3, indicating that they were enriched in bona fide transcriptional units (Figure S1D). Using publicly available RNA-Seq data (SRA accession ERP000016) and cDNAs and ESTs deposited in GenBank, transcript models of long RNA molecules were generated, which were then extensively filtered to exclude those with predicted coding potential or insufficient transcription from the predicted strand. This procedure yielded 567 lincRNA gene annotations giving rise to 691 isoforms (Table S2). Of those, 27 genes (4.8%) were contained within introns of protein-coding genes (14 in the sense and 13 the antisense orientation). Hand curation of a subset of the 567 genes confirmed the specificity of our pipeline, uncovering only a few false-positives resulting from either gaps in the genome assembly or short unannotated coding regions (Table S2). As in mammals (Guttman et al., 2009Guttman M. Amit I. Garber M. French C. Lin M.F. Feldser D. Huarte M. Zuk O. Carey B.W. Cassady J.P. et al.Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals.Nature. 2009; 458: 223-227Crossref PubMed Scopus (3277) Google Scholar, Khalil et al., 2009Khalil A.M. Guttman M. Huarte M. Garber M. Raj A. Rivea Morales D. Thomas K. Presser A. Bernstein B.E. van Oudenaarden A. et al.Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.Proc. Natl. Acad. Sci. USA. 2009; 106: 11667-11672Crossref PubMed Scopus (2359) Google Scholar), lincRNA genes were assigned provisional names based on the closest annotated protein-coding gene (Table S2), except for cases in which vertebrate synteny that involved another nearby gene suggested a more suitable name (e.g., linc-plcb2). For comparison to the zebrafish lincRNAs, we filtered human and mouse lincRNA annotations to remove those overlapping protein-coding genes, pseudogenes or small ncRNA genes, such as microRNA genes (Table S3). Despite this filtering, our curated sets of mammalian lincRNA genes, numbering 2,458 in human and 3,345 in mouse, were larger than our set of zebrafish lincRNA genes, in part because our pipeline for lincRNA discovery was more stringent than those used previously (Ponjavic et al., 2007Ponjavic J. Ponting C.P. Lunter G. Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs.Genome Res. 2007; 17: 556-565Crossref PubMed Scopus (564) Google Scholar, Guttman et al., 2009Guttman M. Amit I. Garber M. French C. Lin M.F. Feldser D. Huarte M. Zuk O. Carey B.W. Cassady J.P. et al.Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals.Nature. 2009; 458: 223-227Crossref PubMed Scopus (3277) Google Scholar, Guttman et al., 2010Guttman, M., Garber, M., Levin, J.Z., Donaghey, J., Robinson, J., Adiconis, X., Fan, L., Koziol, M.J., Gnirke, A., Nusbaum, C., et al. (2010). Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nat. Biotechnol. 28, 503–510.Google Scholar, Jia et al., 2010Jia H. Osak M. Bogu G.K. Stanton L.W. Johnson R. Lipovich L. Genome-wide computational identification and manual annotation of human long noncoding RNA genes.RNA. 2010; 16: 1478-1487Crossref PubMed Scopus (309) Google Scholar, Orom et al., 2010Orom U.A. Derrien T. Beringer M. Gumireddy K. Gardini A. Bussotti G. Lai F. Zytnicki M. Notredame C. Huang Q. et al.Long noncoding RNAs with enhancer-like function in human cells.Cell. 2010; 143: 46-58Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar), in that it required independent experimental evidence for transcriptional initiation, elongation and termination at each locus. In analyzing whole animals our analysis also might have missed many lincRNAs with very restricted expression patterns. Our set of zebrafish lincRNAs shared many characteristics with mammalian lincRNAs. Most (61.7%) were spliced. They averaged 1,951 nt spanning an average of 2.3 exons, were more A/U-rich than coding sequences and 5′UTRs, but less so than 3′UTRs, and resembled 5′UTRs in prevalence of short homopolymers (Figures S1E–S1H). lincRNAs from zebrafish, mouse and human are more likely than protein-coding genes to overlap with repetitive elements, but compared with mammalian lincRNAs, a smaller portion of zebrafish lincRNA sequence was repetitive (Figure S1I). Mammalian lincRNA genes tend to be within < 10 kb of protein-coding genes (Bertone et al., 2004Bertone P. Stolc V. Royce T.E. Rozowsky J.S. Urban A.E. Zhu X. Rinn J.L. Tongprasit W. Samanta M. Weissman S. et al.Global identification of human transcribed sequences with genome tiling arrays.Science. 2004; 306: 2242-2246Crossref PubMed Scopus (870) Google Scholar, Ponjavic et al., 2007Ponjavic J. Ponting C.P. Lunter G. Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs.Genome Res. 2007; 17: 556-565Crossref PubMed Scopus (564) Google Scholar, Jia et al., 2010Jia H. Osak M. Bogu G.K. Stanton L.W. Johnson R. Lipovich L. Genome-wide computational identification and manual annotation of human long noncoding RNA genes.RNA. 2010; 16: 1478-1487Crossref PubMed Scopus (309) Google Scholar). Although zebrafish lincRNA genes also tended to reside near protein-coding genes (empirical p < 0.001 compared to random intergenic regions; Figure S1J), the distances between lincRNA genes and the closest protein-coding genes were similar to the distances between adjacent protein-coding genes (Figure S1J). The closest neighboring protein-coding gene was most likely to appear in a divergent orientation with respect to the lincRNA (Figure S1K). Mammalian lincRNAs have also been reported to be enriched near genes encoding transcription factors and genes involved in nervous system development (Mercer et al., 2008Mercer T.R. Dinger M.E. Sunkin S.M. Mehler M.F. Mattick J.S. Specific expression of long noncoding RNAs in the mouse brain.Proc. Natl. Acad. Sci. USA. 2008; 105: 716-721Crossref PubMed Scopus (934) Google Scholar, Guttman et al., 2009Guttman M. Amit I. Garber M. French C. Lin M.F. Feldser D. Huarte M. Zuk O. Carey B.W. Cassady J.P. et al.Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals.Nature. 2009; 458: 223-227Crossref PubMed Scopus (3277) Google Scholar, Ponjavic et al., 2009Ponjavic J. Oliver P.L. Lunter G. Ponting C.P. Genomic and transcriptional co-localization of protein-coding and long non-coding RNA pairs in the developing brain.PLoS Genet. 2009; 5: e1000617Crossref PubMed Scopus (316) Google Scholar), although these trends are potentially confounded by larger intergenic regions surrounding these genes (Taher and Ovcharenko, 2009Taher L. Ovcharenko I. Variable locus length in the human genome leads to ascertainment bias in functional inference for non-coding elements.Bioinformatics. 2009; 25: 578-584Crossref PubMed Scopus (21) Google Scholar). We tested for GO enrichments in the set of protein-coding genes flanking zebrafish lincRNA loci (Figure S1L). The closest neighbors of zebrafish lincRNAs were significantly more likely to function in transcription-related processes (2.85-fold enrichment, hypergeometric test false-discovery rate [FDR] < 0.05), an enrichment that could not be explained by the larger intergenic regions flanking those genes (empirical p < 0.001). Enrichment for developmental genes was not significant after correction for multiple hypothesis testing or for the sizes of the intergenic regions, with similar trends observed in mouse and human (Figure S1L). We selected a subset of lincRNAs with relatively high expression or conservation and determined their spatial-temporal expression by in situ hybridization at two developmental stages in zebrafish embryos (Table S4). Most tested lincRNAs were expressed in a highly tissue-specific manner (Figure 2A and Figure S2), predominantly in different parts of the central nervous system, although some were expressed in nonneural tissues and cell types, such as the pronephros (linc-cldn7a) and notochord (linc-trpc7). Although our pipeline was expected to miss lincRNAs expressed in very few cells (because their ChIP-Seq signal from entire embryos might not have exceeded background), it did identify many lincRNAs with tissue-specific expression, suggesting diverse and specific roles for these noncoding RNAs.Figure S2Whole-Mount In Situ Hybridizations Using Sense Probes for Selected lincRNAs, Related to Figure 2View Large Image Figure ViewerDownload Hi-res image Download (PPT) We used RNA-Seq data from ten developmental stages and tissues (SRA study ERP000016) to characterize lincRNA expression across zebrafish development. Akin to mammalian lincRNAs (Ponjavic et al., 2009Ponjavic J. Oliver P.L. Lunter G. Ponting C.P. Genomic and transcriptional co-localization of protein-coding and long non-coding RNA pairs in the developing brain.PLoS Genet. 2009; 5: e1000617Crossref PubMed Scopus (316) Google Scholar, Guttman et al., 2010Guttman M. Garber M. Levin J.Z. Donaghey J. Robinson J. Adiconis X. Fan L. Koziol M.J. Gnirke A. Nusbaum C. et al.Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs.Nat. Biotechnol. 2010; 28: 503-510Crossref PubMed Scopus (982) Google Scholar), expression levels of zebrafish lincRNAs were generally lower than those of protein-coding genes (Kolmogorov-Smirnov test, p < 10−15; Figure 2B). Across the ten developmental stages/tissues, expression of lincRNAs tended to correlate with that of their nearest protein-coding neighbors (average Spearman correlation r2 = 0.14, p < 0.001). Correlation of a similar magnitude was observed for adjacent protein-coding genes (r2 = 0.13" @default.
• W2156880688 created "2016-06-24" @default.
• W2156880688 creator A5016728879 @default.
• W2156880688 creator A5043206263 @default.
• W2156880688 creator A5046349314 @default.
• W2156880688 creator A5087589263 @default.
• W2156880688 creator A5088681851 @default.
• W2156880688 date "2011-12-01" @default.
• W2156880688 modified "2023-10-12" @default.
• W2156880688 title "Conserved Function of lincRNAs in Vertebrate Embryonic Development despite Rapid Sequence Evolution" @default.
• W2156880688 cites W1767772592 @default.
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