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

Abstract

BackgroundMicroRNAs 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 FindingsSOLiD 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/SignificanceExtending 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.

A bias-reducing strategy in profiling small RNAs using Solexa

“…We noticed that most of the 42xx and 43xx human miRNAs were missing in our results. These miRNAs were recently predicted from sequences cloned from human embryonic stem cells and neural precursors using the SOLiD platform (Goff et al 2009). We also noticed that most of the 55x to 66x human miRNAs that were cloned with miRAGE were either absent or present at low abundance in our results (Supplemental Table S6; Cummins et al 2006).…”

Prevalence ofPIK3CAmutations and the SNP rs17849079 in Arab breast cancer patients

“…22 Interestingly, the microRNA (miRNA), hsa-miR-4324 (accession #MI0015854), binds to the PIK3CA mRNA sequence at exon 20, which harbors this SNP. 40,41 This miRNA seems to bind only the wild-type allele but not the variant sequence containing this SNP. The functional significance of this could be that miRNA regulation of PIK3CA expression is affected by the presence of this genetic variation.…”

High-performance quantification of mature microRNAs by real-time RT-PCR using deoxyuridine-incorporated oligonucleotides and hemi-nested primers

“…However, due to an additional probe hydrolysis step, TaqMan assays were not compatible with fast thermocycling protocols for rapid detection of miRNAs. Furthermore, with the escalating identification of hundreds of candidate miRNAs by deep sequencing (Bar et al 2008;Goff et al 2009), the design of the TaqMan probe for each of the novel miRNAs is not only cost prohibitive, but also technically challenging, and it faces practical difficulties (Varkonyi-Gasic et al 2007). Attempts have been made to improve miRNA detection without reliance on fluorescent probes (Raymond et al 2005;Shi and Chiang 2005;Sharbati-Tehrani et al 2008); however, these assays usually involved multiple sample processing steps (Shi and Chiang 2005) and suffered from a limited dynamic range of detection (Raymond et al 2005) and/or poor specificity against homologous miRNAs (Raymond et al 2005;Shi and Chiang 2005;Sharbati-Tehrani et al 2008).…”