The RNA Binding Activity of a Ribosome Biogenesis Factor, Nucleophosmin/B23, Is Modulated by Phosphorylation with a Cell Cycle-dependent Kinase and by Association with Its Subtype

Nucleophosmin/B23 is a nucleolar phosphoprotein. It has been shown that B23 binds to nucleic acids, digests RNA, and is localized in nucleolar granular components from which preribosomal particles are transported to cytoplasm. The intracellular localization of B23 is significantly changed during the cell cycle. Here, we have examined the cellular localization of B23 proteins and the effect of mitotic phosphorylation of B23.1 on its RNA binding activity. Two splicing variants of B23 proteins, termed B23.1 and B23.2, were complexed both in vivo and in vitro. The RNA binding activity of B23.1 was impaired by hetero-oligomer formation with B23.2. Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association. Interphase B23.1 was mainly localized in nucleoli, whereas B23.2 and mitotic B23.1, those of which were incapable of binding to RNA, were dispersed throughout the nucleoplasm and cytoplasm, respectively. These results suggest that nucleolar localization of B23.1 is mediated by its ability to associate with RNA

Two Splice Variants of Nopp140 in Drosophila melanogaster

The Nopp140 gene of Drosophila maps within 79A5 of chromosome 3. Alternative splicing yields two variants. DmNopp140 (654 residues) is the sequence homolog of vertebrate Nopp140. Its carboxy terminus is 64% identical to that of the prototypical rat Nopp140. DmNopp140-RGG (688 residues) is identical to DmNopp140 throughout its first 551 residues, but its carboxy terminus contains a glycine/arginine-rich domain that is often found in RNA-binding proteins such as vertebrate nucleolin. Both Drosophila variants localize to nucleoli in Drosophila Schneider II cells and Xenopus oocytes, specifically within the dense fibrillar components. In HeLa cells, DmNopp140-RGG localizes to intact nucleoli, whereas DmNopp140 partitions HeLa nucleoli into phase-light and phase-dark regions. The phase-light regions contain DmNopp140 and endogenous fibrillarin, whereas the phase-dark regions contain endogenous nucleolin. When coexpressed, both Drosophila variants colocalize to HeLa cell nucleoli. Both variants fail to localize to endogenous Cajal bodies in Xenopus oocyte nuclei and in HeLa cell nuclei. Endogenous HeLa coilin, however, accumulates around the periphery of phase-light regions in cells expressing DmNopp140. The carboxy truncation (DmNopp140ΔRGG) also fails to localize to Cajal bodies, but it forms similar phase-light regions that peripherally accumulate endogenous coilin. Conversely, we see no unusual accumulation of coilin in cells expressing DmNopp140-RGG