THE DYNAMIC ORGANIZATION OF THE PERINUCLEOLAR COMPARTMENT IN THE CELL-NUCLEUS

The perinucleolar compartment (PNC) is a unique nuclear structure pref erentially localized at the periphery of the nucleolus. Several small RNAs transcribed by RNA polymerase III (e.g., the Y RNAs, MRP RNA, and RNase P HI RNA) and the polypyrimidine tract binding protein (PTB; hn RNP I) have thus far been identified in the PNC (Ghetti, A., S. Pinol- Roma, W.M. Michael, C. Morandi, and G. Dreyfuss. 1992. Nucleic Acids R es. 20:3671-3678; Matera, A.G., M.R. Frey, K. Margelot, and S.L. Wolin . 1995. J. Cell Biol. 129:1181-1193; Lee, B., A.G. Matera, D.C. Ward, and J. Craft. 1996. Proc. Natl. Acad. Sci. USA. 93: 11471-11476). In t his report, we have further characterized this structure in both fixed and living cells. Detection of the PNC in a large number of human can cer and normal cells showed that PNCs are much more prevalent in cance r cells. Analysis through the cell cycle using immunolabeling with a m onoclonal antibody, SH54, specifically recognizing PTB, demonstrated t hat the PNC dissociates at the beginning of mitosis and reforms at lat e telophase in the daughter nuclei. To visualize the PNC in living cel ls, a fusion protein between PTB and green fluorescent protein (GFP) w as generated. Time lapse studies revealed that the size and shape of t he PNC is dynamic over time. In addition, electron microscopic examina tion in optimally fixed cells revealed that the PNC is composed of mul tiple strands, each measuring similar to 80-180 nm diam. Some of the s trands are in direct contact with the surface of the nucleolus. Furthe rmore, analysis of the sequence requirement for targeting PTB to the P NC using a series of deletion mutants of the GFP-PTB fusion protein sh owed that at least three RRMs at either the COOH or NH2 terminus are r equired for the fusion protein to be targeted to the PNC. This finding suggests that RNA binding may be necessary for PTB to be localized in the PNC.