Fig. 9. hNMD3 associates with nascent 60S ribosomal subunit in both the nucleus and the cytoplasm. (A) Nucleoplasmic and cytoplasmic extracts of oocytes injected with MBP–hNMD3-NESmut fusion protein and labeled with [32P]GTP were fractionated on 10–40% sucrose gradients, and the distributions of newly made rRNAs (top panels) and MBP–hNMD3 (bottom panels) were determined by agarose gel electrophoresis and western blotting with anti-MBP antibodies, respectively. In the experiment shown, the nucleoplasmic extract was prepared from oocytes treated with VSV M protein (an inhibitor of nuclear export; Her et al., 1997) to increase the levels of 40S and 60S export complexes in the nucleoplasm, but both wild-type and NESmut hNMD3 proteins are also associated with nascent 60S subunits in nucleoplasmic extracts of untreated control oocytes [unpublished data; compare with (B)]. (B and C) Nuclear extracts of oocytes injected with wild-type (WT) or NESmut MBP–hNMD3 proteins were immunoprecipitated with anti-MBP antibodies, and the large (B) and small (C) rRNAs of the bound (Ppt) and unbound (Sup) fractions and the total (Tot) extract were analyzed by gel electrophoresis as in Figure 8. M: marker rRNAs as indicated. Note that the nucleoplasmic extracts are devoid of nucleoli, which contain the majority of the 32S, 36S and 40S precursor rRNA, and which are abundant in total nuclear RNAs (Figure 6B).