Figure 4.

ANKHD1 binds to miR-29a-3p, -196a, and -205 via its single KH domain. A, endogenous RIPs for miR-29a-3p, -196a, and -205 were performed using anti-ANKHD1 or IgG control antibodies. The results are presented as fold enrichment over IgG. All experiments were performed three times, and p values of less than 0.05 were considered significant using a Student's t test. B, a full-length FLAG-tagged ANKHD1 construct (FL-WT) was used to generate a truncation mutation, a FLAG-tagged version of ANKHD1 that lacks the ankyrin repeats (KH-WT, 16 kDa). The canonical GXXG loop was mutated to GDDG to produce a FLAG-tagged full-length mutant construct (FL-Mut). C, sequencing was carried out to confirm the mutation in the FL-Mut construct. D and E, RIP pulldowns were performed in RCC4 cells following transfection of either FL-WT or FL-mut or KH-WT constructs. qPCR analysis of either miR-29a-3p (D) or miR196a (E) revealed that these physically interacted with the full-length protein, as well as the KH-only proteins, but were unable to interact with the mutant construct (FL-mut). F, RCC4 were transfected either with an empty vector (EV) or ANKHD1 full-length (FL-WT) or the full-length mutant (FL-Mut), and the cells were stained with pH3 and subjected to flow cytometry to measure mitosis. The experiment was performed three times, and a p value of less than 0.05 was considered as significant using a one-way ANOVA. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.