We previously demonstrated the critical role of RNA polymerase I (Pol I)-associated factor PAF53 in mammalian rRNA transcription. Here, we report the isolation and characterization of another Pol I-associated factor, PAF49. Mouse PAF49 shows striking homology to the human nucleolar protein ASE-1, so that they are considered orthologues. PAF49 and PAF53 were copurified with a subpopulation of Pol I during purification from cell extracts. Physical association of PAF49 with Pol I was confirmed by a coimmunoprecipitation assay. PAF49 was shown to interact with PAF53 through its N-terminal segment. This region of PAF49 also served as the target for TAF(I)48, the 48-kDa subunit of selectivity factor SL1. Concomitant with this interaction, the other components of SL1 also coimmunoprecipitated with PAF49. Specific transcription from the mouse rRNA promoter in vitro was severely impaired by anti-PAF49 antibody, which was overcome by addition of recombinant PAF49 protein. Moreover, overexpression of a deletion mutant of PAF49 significantly reduced pre-rRNA synthesis in vivo. Immunolocalization analysis revealed that PAF49 accumulated in the nucleolus of growing cells but dispersed to nucleoplasm in growth-arrested cells. These results strongly suggest that PAF49/ASE-1 plays an important role in rRNA transcription.
Extracts obtained from mouse cells growth arrested at stationary phase or under serum starvation exhibit no specific rDNA transcription activity. Experiments with mixed transcriptionally active and inactive whole cell extracts (WCE) obtained from rapidly dividing or growth arrested cells, respectively, demonstrate that rRNA synthesis in vitro can be suppressed by a polymerase I transcription inhibitory activity (PIN), present in inactive extracts. This inhibition effect is not related to increased nuciease activity and affects neither the nonspecific Pol I transcription, nor a polymerase II promoter. A comparison of WCE isolated under different growth conditions indicates that PIN changes according to the physiological state of the cell, it reaches a maximal level soon after serum depletion and disappears rapidly when cells are allowed to recover in serum-rich medium. PIN can be clearly demonstrated in WCE but not in nuclear or cytopiasmic extracts and can be also obtained by an additional high salt extraction of nuclei. Furthermore, gel retardation and transcription-in-pallet assays demonstrate that rDNA promoter binding and preinitlatlon complex stability are similar in active and inactive WCE. This indicates that some later stage(s) of rDNA transcription, rather than the preinitiation complex formation, are attenuated by inactive extracts. Analysis of partially fractionated extracts suggests that PIN is not associated with but can be separated from polymerase I.
The molecular mechanism of any tumor marker expression may shed a light on the mechanism of the particular tumorigenesis. This idea in mind, we have been pursuing the mechanism of specific induction of the placental type glutathione transferase (GST-P) gene during hepatocarcinogenesis of the rat. Making use of advanced technologies of molecular biology including proteomic analysis, gene cloning and production of specific transgenic rats etc., we were able to identify the enhancer and the activator proteins responsible for this tumor marker expression. Negative regulatory regions and modulatory proteins were also found. The overview of this long range study and the future outlook of the problem will be discussed. (Communicated by Kumao TOYOSHIMA, M.J.A.)
Ehrlich ascites tumor cells were made permeable to nucleoside triphosphates by treating them with diethylaminoethyl (DEAE)-dextran. Permeable cells incorporated labeled UTP into RNA at a constant rate for at least 30 min at 37°C and 100 min at 25°C, whereas no incorporation was detected without DEAE-dextran treatment. This RNA synthesis is dependent on added nucleoside triphosphates and is affected differentially by different concentrations of α-amanitin. The results of both hybridization and S1-nuclease protection mapping experiments with pulse-labeled RNA synthesized in this system indicate that reinitiation of rRNA transcription takes place from the physiological initiation site in cells made permeable by treatment with DEAE-dextran. By using this system, we examined the kinetics of rRNA synthesis in mouse FM3A cells during inhibition of protein synthesis. The results reflected those previously obtained by analyses of nucleolar RNA synthesis in vivo during protein synthesis inhibition. This permeable cell system should provide a rapid and reproducible assay method for rRNA synthesis in mammalian cells.
