Nucleolar localization of human methionyl-tRNA synthetase and its role in ribosomal RNA synthesis

Human aminoacyl-tRNA synthetases (ARSs) are normally located in cytoplasm and are involved in protein synthesis. In the present work, we found that human methionyl-tRNA synthetase (MRS) was translocated to nucleolus in proliferative cells, but disappeared in quiescent cells. The nucleolar localization of MRS was triggered by various growth factors such as insulin, PDGF, and EGF. The presence of MRS in nucleoli depended on the integrity of RNA and the activity of RNA polymerase I in the nucleolus. The ribosomal RNA synthesis was specifically decreased by the treatment of anti-MRS antibody as determined by nuclear run-on assay and immunostaining with anti-Br antibody after incorporating Br-UTP into nascent RNA. Thus, human MRS plays a role in the biogenesis of rRNA in nucleoli, while it is catalytically involved in protein synthesis in cytoplasm.     

Intranucleolar localization of the RNA polymerase A activity in isolated nuclei of regenerating rat liver

Ultrastructural autoradiography was used to visualize RNA polymerase A activity in parenchymal cell nuclei isolated from normal and regenerating (3, 24, 36 and 48 h after partial hepatectomy) rat liver. High resolution autoradiography showed that the activity of RNA polymerase A which was not inhibited by α-amanitin in a concentration of 0.8 μg/ml, was restricted to the nucleolus. Both the distribution pattern and number of grains were similar in control liver and regenerating liver 3 h after hepatectomy. Twentyfour, 36, and 48 h after hepatectomy nucleoli were enlarged and labeling was distinctly increased. In all experimental groups the activity of RNA polymerase A was located within fibrillar components of the nucleolus. The association of enzyme activity with this component was especially distinct in later stages (36 and 48 h) of liver regeneration. These results suggest that the fibrillar component of the nucleolus contains the active template for ribosomal RNA (rRNA) synthesis in rat liver cell nuclei.     

Effect of the ionic environment on the transcriptional activity of rat liver nucleoli

The localization of various RNA polymerase activities in the nucleoplasm and the nucleolus was investigated in isolated nuclei and in isolated nucleoli by means of a combination of cell fractionation, biochemical analysis and ultrastructural autoradiography. This resulted in four chief conclusions.

1. 1. The two main RNA polymerase activities can be localized clearly in the interphase nucleus: at low ionic strength, the activity is restricted to the nucleolus whereas at high ionic strength the activity is found in both nucleolar and nucleoplasmic regions.

2. 2. The preservation of the ultrastructure of the nuclei at high ionic strength is rather poor so that the physiological meaning of the activity revealed is questionable.

3. 3. Contrary to other reports, the nucleolar activity is significantly enhanced by increasing ammonium sulphate concentration in the presence of Mn²⁺.

4. 4. This increase is probably related to progressive revealing of RNA polymerase B activity within the nucleolus.

     

Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells

The formation of daughter nuclei and the reformation of nucleolar structures was studied after microinjection of antibodies to RNA polymerase I into dividing cultured cells (PtK2). The fate of several nucleolar proteins representing the three main structural subcomponents of the nucleolus was examined by immunofluorescence and electron microscopy. The results show that the RNA polymerase I antibodies do not interfere with normal mitotic progression or the early steps of nucleologenesis, i.e., the aggregation of nucleolar material into prenucleolar bodies. However, they inhibit the telophasic coalescence of the prenucleolar bodies into the chromosomal nucleolar organizer regions, thus preventing the formation of new nucleoli. These prenucleolar bodies show a fibrillar organization that also compositionally resembles the dense fibrillar component of interphase nucleoli. We conclude that during normal nucleologenesis the dense fibrillar component forms from preformed entities around nucleolar organizer regions, and that this association seems to be dependent on the presence of an active form of RNA polymerase I.     

Changes in "template-bound" and "free" RNA polymerase activities in isolated nuclei from Xenopus laevis embryos

Nuclei have been isolated from Xenopus laevis embryos and incubated under conditions allowing RNA synthesis to proceed for more than 3 h. The RNA molecules synthesized on the endogenous template are stable, heterogeneous in size and correspond to the activities of the three RNA polymerases.In these in vitro conditions we have determined the extent of activity of the three RNA polymerases during the embryonic development from blastula to swimming tadpole. Our results on isolated nuclei are in good agreement with the changes in RNA synthesis which take place during normal embryonic development.We have measured both the “template-bound” and the “free” activities of each of the three RNA polymerases during development. Amongst the total RNA polymerase activities engaged on the template, the proportion of polymerase I increases as development proceeds: at the blastula stage, there is practically no RNA polymerase I engaged on the template, whereas in swimming tadpoles, RNA polymerase I amounts to about 90% of the RNA polymerases bound to the DNA. Conversely, RNA polymerase I represents the major part of free RNA polymerases in blastula nuclei.Autoradiography of incubated nuclei shows that, at least in swimming tadpoles nuclei, both “free” and “template-bound” RNA polymerase I are localized in the nucleoli.The evolution of “template-bound” RNA polymerase II activity during development is quite different from that of RNA polymerase I: RNA polymerase II activity represents 75% of engaged polymerase activity in blastulae and only 47% at the swimming tadpoles stage.The results suggest that part of the “free” RNA polymerase I activity might progressively become “template-bound” during embryogenesis.     

Nucleolar localization of ‘template-bound’ RNA polymerase I in nuclei of Xenopus laevis blastulae

This study shows, by means of autoradiography, that RNA polymerase I activity is present in some of the nuclei isolated from Xenopus blastulae. This activity is localized in one or two nucleoli which have a diameter of at least 1.5 μm. The ratio between nucleolar and total nuclear grain counts allows a quantitative estimation of RNA polymerase I activity relative to total RNA polymerase activity at the blastula stage.     

A possible mechanism by which SV40 T-antigen stimulates rRNA synthesis

Binding of SV40 T-antigen to RNA polymerase I could account for the observation that T-antigen stimulates rRNA synthesis and that nucleoli do not stain for T-antigen. Two tests were performed to detect binding: a) RNA polymerase I was isolated and assayed in the presence of T-antigen; polymerase activity was enhanced. b) RNA polymerase I and T-antigen were mixed and then T-antigen complement fixation assays performed, complement fixation was not inhibited.