Reduction in histone mRNA export efficiencies by knockdown of PHAX. (A) U2OS cells were transfected with siPHAX, followed by transfection with plasmids expressing the indicated genes. After 3-h incubation, RNA was extracted from the nuclear and cytoplasmic fractions of the cells, and the indicated mRNA levels were determined by qRT-PCR analysis. (B) U2OS cells were lysed and immunoprecipitation was performed with an anti-PHAX antibody. PHAX-binding RNA levels were determined by qRT-PCR analysis. (C,D) A mixture of 32P-labeled m7G-capped precursor H2A (pre-H2A), precursor ftz mRNA, and U6Δss was injected into the nucleus of Xenopus oocytes either alone or together with an anti-PHAX antibody. RNA was extracted from nuclear (N) and cytoplasmic (C) fractions 2 h after microinjection and analyzed by 8% denaturing PAGE followed by autoradiography. Pre-H2A was processed into mature H2A (shown as processed-H2A) and pre-ftz mRNA was spliced (spliced-ftz mRNA) in the nucleus. Data are the means ± S.D. (n = 3). (*) P < 0.05, (**) P < 0.01.
