[Identification of the protein partners of the human nucleolar protein SURF6 in HeLa cells by GST pull-down assay].

The eukaryotic proteins comprising the SURF6 protein family are evolutionary conservative and housekeeping proteins however, functional roles of human SURF6 have not been studied so far. To shed light to this question in the present work we applied GST pull-down assay and used two proteins fused with GST, namely human GST-SURF6 and the conservative C-terminal domain of mouse Surf6 that has 85% homology with the C-terminus of the human SURF6 conservative domain (GST-Surf6-dom), to identify SURF6-interacting proteins in human HeLa cells. The results obtained showed that GST-SURF6 interacts with several key nucleolar RNA processing factors (B23/nucleophosmin, nucleolin, EBP2), and also with the specific cofactor of RNA polymerase I, protein UBE These results are the first experimental evidences in favor of participation of the human SURF6 protein in ribosome biogenesis, including transcription of rDNA and processing of rRNAs. The same results were obtained, when GST-Surf6-dom was used to pull-down proteins in HeLa cells. In addition, the panel of the GST-Surf6-dom protein partners, which were identified by mass-spectrometry, points to putative interactions of human SURF6 with a number of nuclear and nucleolar, proteins of other functional groups, i.e. to the protein plurifunctionality.

[Cloning, expression, and isolation from Escherichia coli of human protein SURF-6 translationally fused to glutathione S-transferase].

cDNA of human gene Surf-6 (hSutf-6) was amplified and cloned into vector pGEX-2T for the expression in the bacterial system of protein hSURF-6 translationally fused to glutathione S-transferase. The resulting vector is named as pGEX-2T-GST-hSurf-6. Superproducer of chimeric protein GST-hSURF-6 was obtained on the basis of Escherichia coli strain BL21-CodonPlus(DE3)-RIL. Its purification was performed by the affinity chromatography on L-glatathione-sepharose. The proportion of recombinant protein GST-hSURF-6 in the optimized conditions was not less than 15% of the total bacterial protein, and up to 7 mg of the protein was isolated from 1 liter of culture of the producer strain. The final fraction of eluate contained approximately 80% of GST-hSURF-6. The amount and the purity of the isolated protein were sufficient to immunize animals and obtain antibodies. Protein GST-hSURF-6 can also be used as an affinity ligand for revealing protein partners of hSURF-6 in human cells.

[Overexpression of the nucleolar protein SURF-6 in mouse fibroblasts NIH/3T3 leads to stabilisation of intragenic transcribed spacers of the pre-rRNA].

SURF-6 is an evolutionary conserved nucleolar protein that is required for maintenance of cell viability, but its functional significance in mammals still remains illusive. In the present work we examined effects of SURF-6 overexpression in mouse NIH/3T3 fibroblasts transfected with two plasmids. The plasmid pUHrT62-1 encodes a tetracycline-dependant trans-activator, the protein rtTA, the plasmid pBI-SURF6--the genes of EGFP (enhanced green fluorescent protein) and of mouse SURF-6 which expression was controlled by the rtTA-responsive bi-directorial promoter. Western blot analysis showed that the SURF-6 level was severely augmented in cells transfected with pUHrT62-1 and pBI-SURF6 and incubated with the inducer--doxycycline opposed to the transfected but not-induced cells. The increase of SURF-6 was observed in 24 and 48 h after adding the inducer doxycycline. Dot-hybridization of isolated RNA with biotinilated oligonucleotide probes to various regions of mouse primarily pre-rRNA transcripts showed that overexpression of SURF-6 enhanced levels of the second intragenic transcribed spacer ITS2 in about seven folds and of the 5' external transcribed spacer 5'ETS in two folds. Amounts of fragments corresponding to 18S, 5.8S and 28S rRNA remained almost unchanged. These observations for the first time demonstrated that mammalian SURF-6 helps to stabilize or prevents premature cleavage of the pre-rRNA intragenic transcribed spacers, particularly of ITS2, similar to its homologue in S. cerevisiae the protein Rrp14. Today metazoan proteins that play a similar role in ribosome biogenesis, are not described.

Early expression of nucleolar SURF-6 protein in mouse spleen lymphocytes activated for proliferation in vitro.

Using specific antibodies we studied the content of nucleolar SURF-6 protein, which participates in rRNA processing, in mouser spleen lymphocytes activated for proliferation with concanavalin A and compared it with the content of nucleolar nucleophosmin/B23 protein and DNA replication factor PCNA, well-known markers of proliferating cells. Using immunocytochemistry and immunoblotting methods we demonstrate that the concentration of all these proteins increases simultaneously with increasing the proportion of proliferating cells. Unlike nucleophosmin/B23, SURF-6 protein was not revealed in quiescent lymphocyte nucleoli, while the increase of its level in activated lymphocytes preceded elevation of PCNA level. These observations suggest that nucleolar protein SURF-6 can act as a marker of early T lymphocyte activation for proliferation and that it could participate in cell cycle regulation in mammals.

[Properties and functions of a new nucleolar protein, Surf-6, in 3T3 mouse cells].

The localization of the specific protein Surf-6 from nucleoli of eukaryotic cells in mitosis and its sensitivity to the treatment of cells with RNase A and DNase I in situ were studied. It was shown that, in interphase nucleoli of 3T3 mouse cells, Surf-6 is probably associated with RNA and practically is not associated with DNA. In mitosis, Surf-6 appears in forming nucleoli after the known RNA-binding proteins fibrillarin and B23/nucleofozmin, which are involved in the early and late stages of the assembly of ribosomal particles, respectively. These observations and the regularities of migration of early and late proteins of ribosome assembly to nucleoli in the telophase of mitosis led us to the presumption that Surf-6 is involved in the terminal stages of the assembly of ribosomal particles in murine cells. An immunoblot analysis of the Surf-6 content in synchronized 3T3 cells showed for the first time that Surf-6 is present at all stages of the cell cycle but its content markedly decreases when cells enter the G0 period. Conversely, the activation of cells for proliferation is accompanied by an increase in the Surf-6 content. These observations allow one to regard Surf-6 as a marker of the cell proliferative state and suggest its implication in the regulation of the cell cycle. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.

A higher concentration of an antigen within the nucleolus may prevent its proper recognition by specific antibodies.

Transient transfection of HeLa cells with a plasmid encoding the full-length human fibrillarin fused to a green fluorescent protein (GFP) resulted in two major patterns of intensity of the nucleolar labeling for the chimeric protein: weak and strong. Both patterns were maintained in fibrillarin-GFP expressing cells after fixation with formaldehyde. When the fixed fibrillarin-GFP expressing cells were used for immunolabeling with antibodies to fibrillarin, only the nucleoli with a weak GFP-signal became strongly labeled, whereas those with the heavy signals were only lightly stained, if at all. A similar pattern was observed if the cells were immunolabeled with antibodies to GFP. These observations suggest that an increase in antigen accumulation within the nucleolus, which could take place under various physiological or experimental conditions, could prevent the antigen from being recognized by specific antibodies. These results have implications regarding contradictory data on localization of various nucleolar antigens obtained by conventional immunocytochemistry.