(A) Silencing of AC6 3′UTR luciferase reporter by HA-Bicc1 in the absence or presence of ANKS3-Flag or v5-ANKS6 in transfected HEK293T cells. β-Galactosidase was co-transfected for signal normalization. Data represent mean ± SEM of 3 experiments. *p < 0.05, **p < 0.01.
(B) Principle of the MS2-YFP co-localization assay. The 3′end of the Luc-AC6 reporter mRNA was fused to 27 MS2 hairpins, which can bind the MS2 protein fusedtoYFP. CDS, codingsequence;prox, proximal.
(C and D) Localization of the Luc-AC6-MS2327 reporter mRNA and HA-Bicc1 in the absence (C) or presence (D) of ANKS3-Flag in transfected HeLa cells. The MS2-tagged mRNA was imaged by nuclear MS2-YFP fusion protein that is retained in the cytoplasm when bound to the MS2 RNA hairpins. HA-Bicc1 and ANKS3-Flag were detected by immunofluorescent staining using anti-HA or anti-Flag antibodies. Scale bars, 5 μm.