Nucleolar clustering of dispersed tRNA genes - PubMed (original) (raw)

Nucleolar clustering of dispersed tRNA genes

Martin Thompson et al. Science. 2003.

Abstract

Early transfer RNA (tRNA) processing events in Saccharomyces cerevisiae are coordinated in the nucleolus, the site normally associated with ribosome biosynthesis. To test whether spatial organization of the tRNA pathway begins with nucleolar clustering of the genes, we have probed the subnuclear location of five different tRNA gene families. The results show that tRNA genes, though dispersed in the linear genome, colocalize with 5S ribosomal DNA and U14 small nucleolar RNA at the nucleolus. Nucleolar localization requires tRNA gene transcription-complex formation, because inactivation of the promoter at a single locus removes its nucleolar association. This organization of tRNA genes must profoundly affect the spatial packaging of the genome and raises the question of whether gene types might be coordinated in three dimensions to regulate transcription.

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Figures

Fig. 1

Fig. 1

Nucleolar localization of tRNA genes. Fluorescent oligonucleotide probes were annealed to individual tRNA gene families (Leu, Gly, Gln, Lys, and Glu) or to the telomeric repeats. Each family of tRNA genes has 9 to 16 members that are dispersed in the linear genome map (28) (fig. S1). The Cy3-labeled probes to the tRNA genes and telomeres are red, and the Oregon Green 488 probe to the 5_S_ rRNA gene is green. 4′,6′-diamidino-2-phenylindole (DAPI) staining of the nucleoplasm is shown as blue. Most of the tRNA gene signal overlaps the nucleolar 5_S_ rRNA genes. In contrast, the telomeric repeat probe stains the nuclear periphery and nucleoplasm. Cells shown are representative of multiple experiments.

Fig. 2

Fig. 2

tRNA gene localization depends on Pol III complex formation. The SUP53 tRNA gene locus on chromosome III was probed by replacing the neighboring LEU2 coding region with URA3 coding sequences and simultaneously probing fixed cells with 14 fluorescent oligonucleotides (red) complementary to the non-RNA strand of URA3. Nucleoli were identified with a fluorescent oligonucleotide to U14 snoRNA (green), and the nucleoplasm is stained with DAPI (blue). The endogenous URA3 coding sequences are deleted from chromosome V in these strains. As a negative control, a strain in which the URA3 sequences were deleted by replacement with a kanamycin resistance gene (Kan) showed no signal with the URA3 probes (lowest panel). SUP53 frequently localized in or showed some overlap with the nucleolus when transcriptionally active (52% out of 440 cells imaged), but did so less often when the promoter was mutated to be inactive (13% out of 715 cells imaged).

Fig. 3

Fig. 3

Pol I affects tRNA gene clusters. The positions (red) of the tRNALeu gene family and its pre-tRNA transcripts were probed (17) in a strain with a deleted, nonessential subunit of Pol I (Δ_rpa49_) and compared with the parental wild-type strain (WT). Nucleoli are marked by a probe to the U14 snoRNA (green) and nucleoplasm is stained with DAPI (blue) in all panels.

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