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

Figure 3

Ago2 IP-selected microRNAs.

A. Hierarchical clustering dendrogram showing Pearson correlation between ESC and NSC stages of four hESC lines: H1, HSF1, HSF6, and RG7. Expression was determined by counting occurrences based on the list of 609 known or 146 predicted microRNAs found to be Ago2 IP enriched. Counts were normalized to the total number of sequences per sample. For both known microRNAs (top) and predicted microRNAs (bottom), the primary distinction between samples is based on cell stage. This demonstrates that each group of microRNAs is uniformly expressed in a group of biologically distinct cell lines. B. Pearson correlation tables of microRNA expression across development. Using the list of 609 known microRNAs (top) or 146 predicted microRNAs (bottom) selected from the Ago2 IP enriched lists, expression levels were determined by counting sequences and correcting for total numbers of sequences per sample. Known microRNA expression levels (top) distinguished all three adult tissues from a pluripotent group (RG7 ESC and iPS cells obtained from WiCell) and also from a neural lineage group (RG7 NSC, RG7 NRP, and iPS-derived NSC). However, predicted microRNAs included the adult brain sample in a cluster containing iPS NSC and, to a lesser extent, RG7 NSC and NRP. C. The same dataset was K-means clustered to isolate expression patterns. Based on minimizing the mean sum of squares fit (Figure S6), k = 11 clusters were selected. Shown here are the cluster means±SEM, color-coded to match the complete plotting of all 755 clustered known and new microRNAs (Figure S7). D. Conservation of predicted microRNAs determined using the novel SiPhy metric ω; substitution rate relative to an ancestral repeat-based neutral evolution model (grey dashed line) [45]. Known microRNAs (black) exhibit two distinct peaks with the larger one (left) having relatively high conservation across 29 mammalian species. The 818 miRDeep-predicted microRNAs (red), in contrast, overlapped with the less conserved fraction of the known microRNAs. A small portion of the 146 Ago2 IP selected microRNAs (blue) peaked as a conserved-sequence group but the majority aligned with the non-conserved microRNAs.

Figure 3

doi: https://doi.org/10.1371/journal.pone.0007192.g003