Pre-miRNA Loop Nucleotides Control the Distinct Activities of mir-181a-1 and mir-181c in Early T Cell Development (original) (raw)
Figure 2
Effects of the mutations in the stem region on mir-181a-1 activity in promoting DP cell development.
(A) Scanning mutations in the stem region of the mir-181a-1 gene. Two nucleotides (2-nt mutants, shaded yellow) or a stretch of nucleotides (segment mutants, green) in the mature miRNA region are altered (shown in red). Nucleotides are altered to disrupt the potential base pairing to target genes. Compensatory mutations are also generated on the miR* strand to maintain the secondary structure of the pre-miRNAs. (B) Expression and processing of wild-type mir-181a-1 and stem mutants. Specific probes that perfectly match the mature miR-181a and each of its mutant forms were used in hybridization to determine the expression of mature miR-181a and its stem mutant forms. (C) The effects of mir-181a-1 and its stem mutants on DP cell development. Normalized data from 3–5 independent T cell assays (each with 12 independent replicates, total 36–60 replicates) are pooled and graphed in the distribution box plots to summarize the distribution of the relative activities of mir-181a-1 (shaded grey), the 2-nt mutants (shaded yellow), and the segment mutants (shaded green) in DP cell development. Mann-Whitney Rank Sum Tests were performed to determine whether the activities of individual 2-nt mutants were statistically different from those of the control vector (red *, p<0.0001) and the mir-181a-1 vector (black *, p<0.0001; **, p<0.05) (Table S1 for statistics). A representative OP9-DL1 stromal co-culture assay without normalization (12 independent replicates for each constructs) is also shown (Fig. S5).