Overexpression of the nucleolar protein SURF-6 in mouse NIH/3T3 fibroblasts stabilizes pre-rRNA intragenic transcribed spacers

Abstract

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.