Ago2 Immunoprecipitation Identifies Predicted MicroRNAs in Human Embryonic Stem Cells and Neural Precursors

Background MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be undiscovered microRNAs expressed early in stem cell differentiation. Methodology/Principal Findings SOLiD ultra-deep sequencing identified >107 unique small RNAs from human embryonic stem cells (hESC) and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs. Conclusions/Significance Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation.

[1]  Mike J. Mason,et al.  Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures. , 2009, Cell stem cell.

[2]  E. Cuppen,et al.  Limitations and possibilities of small RNA digital gene expression profiling , 2009, Nature Methods.

[3]  Wen-Hsiung Li,et al.  Lowly expressed human microRNA genes evolve rapidly. , 2009, Molecular biology and evolution.

[4]  Xiaohui Xie,et al.  Identifying novel constrained elements by exploiting biased substitution patterns , 2009, Bioinform..

[5]  G. Pan,et al.  MicroRNA-145 Regulates OCT4, SOX2, and KLF4 and Represses Pluripotency in Human Embryonic Stem Cells , 2009, Cell.

[6]  Chung-I Wu,et al.  Evolution under canalization and the dual roles of microRNAs: a hypothesis. , 2009, Genome research.

[7]  Xiaozhong Wang,et al.  Essential and overlapping functions for mammalian Argonautes in microRNA silencing. , 2009, Genes & development.

[8]  Haifan Lin,et al.  MicroRNAs: key regulators of stem cells , 2009, Nature Reviews Molecular Cell Biology.

[9]  M. Zavolan,et al.  Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. , 2008, RNA.

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

[11]  W. L. Ruzzo,et al.  MicroRNA Discovery and Profiling in Human Embryonic Stem Cells by Deep Sequencing of Small RNA Libraries , 2008, Stem cells.

[12]  Simon Kasif,et al.  Genomewide Analysis of PRC1 and PRC2 Occupancy Identifies Two Classes of Bivalent Domains , 2008, PLoS genetics.

[13]  D. Bartel,et al.  The impact of microRNAs on protein output , 2008, Nature.

[14]  Leping Li,et al.  Oct4/Sox2-Regulated miR-302 Targets Cyclin D1 in Human Embryonic Stem Cells , 2008, Molecular and Cellular Biology.

[15]  T. Rana,et al.  Evolutionary Emergence of microRNAs in Human Embryonic Stem Cells , 2008, PloS one.

[16]  Steven M. Johnson,et al.  A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning. , 2008, Genome research.

[17]  Gunter Meister,et al.  A multifunctional human Argonaute2-specific monoclonal antibody. , 2008, RNA.

[18]  S. Ranade,et al.  Stem cell transcriptome profiling via massive-scale mRNA sequencing , 2008, Nature Methods.

[19]  Daniel Herschlag,et al.  Systematic Identification of mRNAs Recruited to Argonaute 2 by Specific microRNAs and Corresponding Changes in Transcript Abundance , 2008, PloS one.

[20]  Ronny Lorenz,et al.  The Vienna RNA Websuite , 2008, Nucleic Acids Res..

[21]  N. Rajewsky,et al.  Discovering microRNAs from deep sequencing data using miRDeep , 2008, Nature Biotechnology.

[22]  Philip C. J. Donoghue,et al.  MicroRNAs and the advent of vertebrate morphological complexity , 2008, Proceedings of the National Academy of Sciences.

[23]  L. Goff,et al.  MicroRNA expression pattern of undifferentiated and differentiated human embryonic stem cells. , 2007, Stem cells and development.

[24]  Zhiping P. Pang,et al.  Integrative genomic and functional analyses reveal neuronal subtype differentiation bias in human embryonic stem cell lines , 2007, Proceedings of the National Academy of Sciences.

[25]  G. Churchill,et al.  Characterization of human embryonic stem cell lines by the International Stem Cell Initiative , 2007, Nature Biotechnology.

[26]  M. Zhan,et al.  Whole Genome Analysis of Human Neural Stem Cells Derived from Embryonic Stem Cells and Stem and Progenitor Cells Isolated from Fetal Tissue , 2007, Stem cells.

[27]  Bing Su,et al.  Rapid evolution of an X-linked microRNA cluster in primates. , 2007, Genome research.

[28]  B. Winblad,et al.  Neurogenic neuroepithelial and radial glial cells generated from six human embryonic stem cell lines in serum‐free suspension and adherent cultures , 2007, Glia.

[29]  I. King Jordan,et al.  A Family of Human MicroRNA Genes from Miniature Inverted-Repeat Transposable Elements , 2007, PloS one.

[30]  Rudolf Jaenisch,et al.  DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal , 2007, Nature Genetics.

[31]  C Anthony Blau,et al.  A Comparison of NIH‐Approved Human ESC Lines , 2006, Stem cells.

[32]  R. Place,et al.  Small dsRNAs induce transcriptional activation in human cells , 2006, Proceedings of the National Academy of Sciences.

[33]  Edwin Cuppen,et al.  Diversity of microRNAs in human and chimpanzee brain , 2006, Nature Genetics.

[34]  Eugene Berezikov,et al.  Many novel mammalian microRNA candidates identified by extensive cloning and RAKE analysis. , 2006, Genome research.

[35]  Yvonne Tay,et al.  A Pattern-Based Method for the Identification of MicroRNA Binding Sites and Their Corresponding Heteroduplexes , 2006, Cell.

[36]  Mitchell D. Probasco,et al.  Feeder-independent culture of human embryonic stem cells , 2006, Nature Methods.

[37]  Stijn van Dongen,et al.  miRBase: microRNA sequences, targets and gene nomenclature , 2005, Nucleic Acids Res..

[38]  D. Rokhsar,et al.  Evidence for a microRNA expansion in the bilaterian ancestor , 2006, Development Genes and Evolution.

[39]  Christoph Flamm,et al.  The expansion of the metazoan microRNA repertoire , 2006, BMC Genomics.

[40]  Z. Mourelatos,et al.  Human mitochondrial tRNAMet is exported to the cytoplasm and associates with the Argonaute 2 protein. , 2005, RNA.

[41]  K. Lindblad-Toh,et al.  Systematic discovery of regulatory motifs in human promoters and 3′ UTRs by comparison of several mammals , 2005, Nature.

[42]  C. Burge,et al.  Conserved Seed Pairing, Often Flanked by Adenosines, Indicates that Thousands of Human Genes are MicroRNA Targets , 2005, Cell.

[43]  R. Aharonov,et al.  Identification of hundreds of conserved and nonconserved human microRNAs , 2005, Nature Genetics.

[44]  J. M. Thomson,et al.  Argonaute2 Is the Catalytic Engine of Mammalian RNAi , 2004, Science.

[45]  S. Moon,et al.  Human embryonic stem cells express a unique set of microRNAs. , 2004, Developmental biology.

[46]  Thomas Tuschl,et al.  Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. , 2004, RNA.

[47]  V. Ambros,et al.  Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation , 2004, Genome Biology.

[48]  P. Sharp,et al.  Embryonic stem cell-specific MicroRNAs. , 2003, Developmental cell.

[49]  G. Ruvkun,et al.  A uniform system for microRNA annotation. , 2003, RNA.

[50]  Austin G Smith,et al.  Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture , 2003, Nature Biotechnology.

[51]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[52]  M S Waterman,et al.  Identification of common molecular subsequences. , 1981, Journal of molecular biology.