XB-IMG-147630
Xenbase Image ID: 147630
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Fig. 3. Validation of the interaction between miR-26 and the smad1 3′UTR. (A) Sequences for miR-26a, and the miR-26 binding sites within the Smad1A and Smad1B 3′UTRs. The seed sequence in miR-26 and the corresponding seed-binding sequences in the 3′UTRs are shown in bold. The position of the miR-26 TPMO is represented by the blue line. The pLuc-Smad1 3′UTR reporter construct (Luc-Smad1, or PLS) is also shown. For luciferase assays shown in B, D, and E, luciferase readings are normalized to those of control samples (which are set to 1) and presented as “relative luc activity”. (B) Luciferase assay comparing activities of either luciferase alone (LUC) or the Luc-Smad1 reporter (PLS) in the presence of either the target protector (TPMO) or a 5-base mispair (mis-TPMO). Embryos were injected with the plasmids and MOs and lysed at st. 10.5. (C) Sequences of PLS and PLS-MUT in the miR26 seed-binding region; PLS-MUT carries 4 mutations across this region. Sequences of miR-26 and the mutated miR-26 that carries complementary mutations across the seed sequence. (D) Luciferase activity from pLuc-Smad1 3′UTR (PLS) in which 4 bases are mutated across the miR-26 seed sequence (PLS-mut). (E) Effects of complementary mutations in the seed-binding sequence of miR-26. In the presence of miR-26, luciferase activity is higher in embryos injected with PLS-mut than with PLS (at left). A mutated version of miR-26 (mtt-26) that is complementary to the mutations in PLS-mut significantly inhibits luciferase activity in embryos injected with PLS-mut (right). miRs were injected as duplexed oligoribonucleotides. For PLS, results with mtt-26 are normalized to those with mir-26; for PLS-mut, results with miR-26 are normalized to those with mtt-26, which is complementary to PLS-mut. N≥3 independent experiments for each panel. ⁎Indicates statistical significance (p<0.05); ⁎⁎, very significant (p<0.01); ⁎⁎⁎, extremely significant (p<0.001). Image published in: Liu C et al. (2016) Copyright © 2016. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |