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

Rutgers Stem Cell Research Center, Rutgers University, Piscataway, New Jersey, United States of America.
PLoS ONE (Impact Factor: 3.23). 09/2009; 4(9):e7192. DOI: 10.1371/journal.pone.0007192
Source: PubMed


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.
SOLiD ultra-deep sequencing identified >10(7) 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.
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.

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    • "High-throughput sequencing is not only able to reveal the expression profiles of known miRNAs, but also is able to discover new miRNAs that have not been recorded previously in any databases, in particular the miRBase repository. Small RNA sequencing has been used to carry out research on various types of stem cells, including embryonic stem cells [24–27], hematopoietic stem cells [28], and neural precursor cells [24]. Novel miRNAs have also been identified using smRNA-seq during neural differentiation of embryonic stem cells [27] and during endothelial differentiation [29]. "
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    • "MiRNAs regulate multiple cellular processes including embryonic stem cell (ESC) self-renewal [11] and neural differentiation [12]–[17]. Previous work identifying novel miRNAs and their expression profiles has established a distinct subset of miRNAs with enriched or specific expression in neural tissues and neural precursors [18]–[20]. Brain-enriched miRNAs such as miR-9, miR-124a, miR-125, and numerous others are induced in primary neural tissues and differentiating primary neurons [20]–[22]. Conversely, several ESC specific miRNAs are down-regulated during retinoic acid-induced differentiation of neuronal precursor cells [12] consistent with the hypothesis that miRNAs are likely to be key regulators of neural differentiation. "
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