<i>mir-181ab1</i> controls the negative feedbacks in NOTCH1 and pre-TCR pathways during normal thymocyte development.

<p>(A) The coding regions of individual candidate targets, which lack all or a majority of predicted miR-181a binding sites, were expressed in thymic progenitors, and their effects on DP cell development were determined by using the OP9-DL1 assay (box plots, n = 12, representative analysis of three independent experiments shown). (B) Wild-type and mutant (red) miR-181a target sites in the <i>Nrarp</i> 3′UTR (T1, T2, T3) and the predicted base pairings between mouse <i>Nrarp</i> and miR-181a. (C) Functional regulation of <i>Nrarp</i> by miR-181a during early T cell development. The <i>Nrarp</i> gene with a wild-type full-length UTR (FL<i>^wt</i>) or a full-length UTR with the miR-181a-binding site mutated (FL<i>^mut</i>) were ectopically expressed in thymic progenitors and tested for effects on early thymocyte development (box plots, n = 48; replicates pooled from four independent experiments). (D) miR-181a-sensitive (ORF+3′UTR) and miR-181a-insensitive (ORF only) phosphatases were ectopically expressed in thymic progenitors and cultured over OP9-DL1 stromal cells to examine their effects on DP development (*, <i>p</i><0.0001). (E) <i>Nrarp</i> shRNAs suppress expression of Nrarp protein as determined by western blot analyses. (F) Effects of <i>Nrarp</i> shRNAs on DP cell development in the OP9-DL1 assay (*, <i>p</i><0.05). (G) Effects of shRNAs targeting <i>SHP1</i>, <i>SHP2</i>, <i>DUSP5</i> or <i>DUSP6</i> on DP cell development in the OP9-DL1 assay. Data in (F) and (G) are displayed as a relative change in % DP in box plots (n = 12, representative of three independent experiments, *, <i>p</i><0.0001). (H) Schematic diagram of the mechanism by which miR-181a may regulate normal T cell development.