SNORD-host RNA Zfas1 is a regulator of mammary development and a potential marker for breast cancer.

Long noncoding RNAs (lncRNAs) are increasingly recognized to play major regulatory roles in development and disease. To identify novel regulators in breast biology, we identified differentially regulated lncRNAs during mouse mammary development. Among the highest and most differentially expressed was a transcript (Zfas1) antisense to the 5' end of the protein-coding gene Znfx1. In vivo, Zfas1 RNA is localized within the ducts and alveoli of the mammary gland. Zfas1 intronically hosts three previously undescribed C/D box snoRNAs (SNORDs): Snord12, Snord12b, and Snord12c. In contrast to the general assumption that noncoding SNORD-host transcripts function only as vehicles to generate snoRNAs, knockdown of Zfas1 in a mammary epithelial cell line resulted in increased cellular proliferation and differentiation, while not substantially altering the levels of the SNORDs. In support of an independent function, we also found that Zfas1 is extremely stable, with a half-life >16 h. Expression analysis of the SNORDs revealed these were expressed at different levels, likely a result of distinct structures conferring differential stability. While there is relatively low primary sequence conservation between Zfas1 and its syntenic human ortholog ZFAS1, their predicted secondary structures have similar features. Like Zfas1, ZFAS1 is highly expressed in the mammary gland and is down-regulated in breast tumors compared to normal tissue. We propose a functional role for Zfas1/ ZFAS1 in the regulation of alveolar development and epithelial cell differentiation in the mammary gland, which, together with its dysregulation in human breast cancer, suggests ZFAS1 as a putative tumor suppressor gene.

[1]  A. Long,et al.  A human cell line from a pleural effusion derived from a breast carcinoma. , 1973, Journal of the National Cancer Institute.

[2]  M. Radu,et al.  Establishment and characterization of a cell line of human breast carcinoma origin. , 1979, European journal of cancer.

[3]  T. Mosmann Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[4]  Santa Jeremy Ono,et al.  A novel cysteine-rich sequence-specific DNA-binding protein interacts with the conserved X-box motif of the human major histocompatibility complex class II genes via a repeated Cys-His domain and functions as a transcriptional repressor , 1994, The Journal of experimental medicine.

[5]  L. Hennighausen,et al.  Mammary epithelial cells undergo secretory differentiation in cycling virgins but require pregnancy for the establishment of terminal differentiation. , 1995, Development.

[6]  L. Hennighausen,et al.  Think globally, act locally: the making of a mouse mammary gland. , 1998, Genes & development.

[7]  W. Filipowicz,et al.  The Host Gene for Intronic U17 Small Nucleolar RNAs in Mammals Has No Protein-Coding Potential and Is a Member of the 5′-Terminal Oligopyrimidine Gene Family , 1998, Molecular and Cellular Biology.

[8]  G. Olsen,et al.  CRITICA: coding region identification tool invoking comparative analysis. , 1999, Molecular biology and evolution.

[9]  J. Hickman,et al.  Developmental regulation of Bcl-2 family protein expression in the involuting mammary gland. , 1999, Journal of cell science.

[10]  A. Hüttenhofer,et al.  RNomics: an experimental approach that identifies 201 candidates for novel, small, non‐messenger RNAs in mouse , 2001, The EMBO journal.

[11]  L. Hennighausen,et al.  Signaling pathways in mammary gland development. , 2001, Developmental cell.

[12]  S. P. Fodor,et al.  Large-Scale Transcriptional Activity in Chromosomes 21 and 22 , 2002, Science.

[13]  R. Raggiaschi,et al.  Dome formation in cell cultures as expression of an early stage of lactogenic differentiation of the mammary gland , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[14]  L. Chodosh,et al.  Functional microarray analysis of mammary organogenesis reveals a developmental role in adaptive thermogenesis. , 2002, Molecular endocrinology.

[15]  D. Haussler,et al.  Ultraconserved Elements in the Human Genome , 2004, Science.

[16]  R. Myers,et al.  An abundance of bidirectional promoters in the human genome. , 2003, Genome research.

[17]  C. Perou,et al.  Cell-Type-Specific Responses to Chemotherapeutics in Breast Cancer , 2004, Cancer Research.

[18]  Jürgen Brosius,et al.  Waste not, want not--transcript excess in multicellular eukaryotes. , 2005, Trends in genetics : TIG.

[19]  D. McNeel,et al.  Antigen-Specific IgG Elicited in Subjects with Prostate Cancer Treated with Flt3 Ligand , 2005, Journal of immunotherapy.

[20]  S. Chuang,et al.  Identification and characterization of a novel gene Saf transcribed from the opposite strand of Fas. , 2005, Human molecular genetics.

[21]  S. Salzberg,et al.  The Transcriptional Landscape of the Mammalian Genome , 2005, Science.

[22]  Wayne Tam,et al.  Accumulation of miR-155 and BIC RNA in human B cell lymphomas. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[23]  K. Williams,et al.  Dendritic BC1 RNA in translational control mechanisms , 2005, The Journal of cell biology.

[24]  Liang-Hu Qu,et al.  Genome-wide analyses of two families of snoRNA genes from Drosophila melanogaster, demonstrating the extensive utilization of introns for coding of snoRNAs. , 2005, RNA.

[25]  K. Blazek,et al.  Transcriptional changes underlying the secretory activation phase of mammary gland development. , 2005, Molecular endocrinology.

[26]  T. Hughes,et al.  A systematic search for new mammalian noncoding RNAs indicates little conserved intergenic transcription , 2005, BMC Genomics.

[27]  P. Stadler,et al.  Mapping of conserved RNA secondary structures predicts thousands of functional noncoding RNAs in the human genome , 2005, Nature Biotechnology.

[28]  Brian S. Clark,et al.  The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator. , 2006, Genes & development.

[29]  J. Mattick,et al.  Experimental validation of the regulated expression of large numbers of non-coding RNAs from the mouse genome. , 2005, Genome research.

[30]  C. Bult,et al.  Discrimination of Non-Protein-Coding Transcripts from Protein-Coding mRNA , 2006, RNA biology.

[31]  M. Gaitanou,et al.  BM88 Is a Dual Function Molecule Inducing Cell Cycle Exit and Neuronal Differentiation of Neuroblastoma Cells via Cyclin D1 Down-regulation and Retinoblastoma Protein Hypophosphorylation* , 2006, Journal of Biological Chemistry.

[32]  Joaquín Dopazo,et al.  BABELOMICS: a systems biology perspective in the functional annotation of genome-scale experiments , 2006, Nucleic Acids Res..

[33]  Sin Lam Tan,et al.  Complex Loci in Human and Mouse Genomes , 2006, PLoS genetics.

[34]  Liang-Hu Qu,et al.  snoSeeker: an advanced computational package for screening of guide and orphan snoRNA genes in the human genome , 2006, Nucleic acids research.

[35]  C. Ponting,et al.  Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs. , 2007, Genome research.

[36]  Thomas D. Schmittgen,et al.  Ultraconserved regions encoding ncRNAs are altered in human leukemias and carcinomas. , 2007, Cancer cell.

[37]  William Stafford Noble,et al.  Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project , 2007, Nature.

[38]  P. Stadler,et al.  RNA Maps Reveal New RNA Classes and a Possible Function for Pervasive Transcription , 2007, Science.

[39]  A. Dion,et al.  A human breast tumor cell line (BT-474) that supports mouse mammary tumor virus replication , 1979, In Vitro.

[40]  T. Gingeras,et al.  Genome-wide transcription and the implications for genomic organization , 2007, Nature Reviews Genetics.

[41]  K. Struhl Transcriptional noise and the fidelity of initiation by RNA polymerase II , 2007, Nature Structural &Molecular Biology.

[42]  M. Obregon,et al.  Gene expression from the imprinted Dio3 locus is associated with cell proliferation of cultured brown adipocytes. , 2007, Endocrinology.

[43]  Paulo P. Amaral,et al.  Noncoding RNA in development , 2008, Mammalian Genome.

[44]  N. Rajewsky,et al.  A human snoRNA with microRNA-like functions. , 2008, Molecular cell.

[45]  Tim R. Mercer,et al.  Differentiating Protein-Coding and Noncoding RNA: Challenges and Ambiguities , 2008, PLoS Comput. Biol..

[46]  Paulo P. Amaral,et al.  Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation. , 2008, Genome research.

[47]  Michael Q. Zhang,et al.  Combinatorial patterns of histone acetylations and methylations in the human genome , 2008, Nature Genetics.

[48]  J. French,et al.  Dynamic interactions between the promoter and terminator regions of the mammalian BRCA1 gene , 2008, Proceedings of the National Academy of Sciences.

[49]  S. Sunkin,et al.  Specific expression of long noncoding RNAs in the mouse brain , 2008, Proceedings of the National Academy of Sciences.

[50]  M. Mourtada-Maarabouni,et al.  GAS5, a non-protein-coding RNA, controls apoptosis and is downregulated in breast cancer , 2009, Oncogene.

[51]  Paulo P. Amaral,et al.  Pervasive transcription of the eukaryotic genome: functional indices and conceptual implications. , 2009, Briefings in functional genomics & proteomics.

[52]  J. Mattick,et al.  Long noncoding RNAs in neuronal-glial fate specification and oligodendrocyte lineage maturation , 2010, BMC Neuroscience.

[53]  Paulo P. Amaral,et al.  Complex architecture and regulated expression of the Sox2ot locus during vertebrate development. , 2009, RNA.

[54]  Paulo P. Amaral,et al.  MEN epsilon/beta nuclear-retained non-coding RNAs are up-regulated upon muscle differentiation and are essential components of paraspeckles. , 2009, Genome research.

[55]  D. Spector,et al.  Long noncoding RNAs: functional surprises from the RNA world. , 2009, Genes & development.

[56]  J. Mattick,et al.  Small RNAs derived from snoRNAs. , 2009, RNA.

[57]  J. Mattick,et al.  Genome-Wide Identification of Long Noncoding RNAs in CD8+ T Cells1 , 2009, The Journal of Immunology.

[58]  J. Mattick,et al.  Long non-coding RNAs: insights into functions , 2009, Nature Reviews Genetics.

[59]  B. Montanini,et al.  Eukaryotic snoRNAs: a paradigm for gene expression flexibility. , 2009, Genomics.

[60]  Lennart Martens,et al.  A guide to the Proteomics Identifications Database proteomics data repository , 2009, Proteomics.

[61]  Michael F. Lin,et al.  Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals , 2009, Nature.

[62]  Wei Zhou,et al.  Implication of snoRNA U50 in human breast cancer. , 2009, Journal of genetics and genomics = Yi chuan xue bao.

[63]  G. Chrousos,et al.  Noncoding RNA Gas5 Is a Growth Arrest– and Starvation-Associated Repressor of the Glucocorticoid Receptor , 2010, Science Signaling.

[64]  A. Gabory,et al.  The H19 locus: Role of an imprinted non‐coding RNA in growth and development , 2010, BioEssays : news and reviews in molecular, cellular and developmental biology.

[65]  J. Rinn,et al.  Large non-coding RNAs: missing links in cancer? , 2010, Human molecular genetics.

[66]  J. Mattick,et al.  Protein-coding and non-coding gene expression analysis in differentiating human keratinocytes using a three-dimensional epidermal equivalent , 2010, Molecular Genetics and Genomics.

[67]  John S. Mattick,et al.  lncRNAdb: a reference database for long noncoding RNAs , 2010, Nucleic Acids Res..