Molecular cloning and gene expression of a fibrillarin homolog of tobacco BY-2 cells.

Fibrillarin is known to play an important role in precursor ribosomal RNA processing and ribosome assembly. The present study describes a fibrillarin homolog gene isolated from tobacco BY-2 cells and its expression during the cell cycle. The cDNA for a fibrillarin homolog, named NtFib1, was first cloned in Nicotiana tabacum with degenerate primers. It encodes 314 amino acids and the deduced amino acid sequence has some highly conserved functional domains, such as the glycine and arginine-rich (GAR) domain for nucleolar localization and the RNA-binding motif. The C-terminal region is highly conserved and has 7 beta-sheets and 7 alpha-helices which are peculiar to fibrillarin. Thus, it is suggested that the fibrillarin homolog of this plant species functions in the same way as the fibrillarin already known from human and yeast cells. Northern blot analysis of BY-2 cells synchronized with aphidicolin or a combination of aphidicolin and propyzamide showed that the histone H4 gene was specifically expressed in the S phase but NtFib1 mRNA remained at high levels during the cell cycle. Examination of the localization of NtFib1 protein tagged with green-fluorescent protein (GFP) suggested that some persisting in the mitotic apparatus was eventually incorporated into reconstructed nucleoli in late telophase. Newly synthesized GFP-tagged NtFib1 protein in the cytoplasm was added to the recycled protein in early mitosis. Highly concentrated actinomycin D completely inhibited the transcription of genes coding for rRNA (rDNA) but did not significantly suppress the amount of either NtFib1 mRNA or protein, although the NtFib1 protein was reversibly dislocated from nucleoli. Although hypoxic shock completely prohibited rDNA transcription, NtFib1 mRNA remained at the same level as in the control experiment, even after the 4 h treatment. These results indicate that the transcription of NtFib1 mRNA is not related to rDNA transcription and NtFib1 mRNA is resistant to disrupting factors during the cell cycle.