Nucleolar localization of early tRNA processing

Abstract

There is little information as to the location of early tRNA biosynthesis. Using fluorescent in situ hybridization in the budding yeast, Saccharomyces cerevisiae, examples of nuclear pre-tRNAs are shown to reside primarily in the nucleoli. We also probed the RNA subunit of RNase P. The majority of the signal from RNase P probes was nucleolar, with less intense signals in the nucleoplasm. These results demonstrate that a major portion of the tRNA processing pathway is compartmentalized in nucleoli with rRNA synthesis and ribosomal assembly. The spatial juxtaposition suggests the possibility of direct coordination between tRNA and ribosome biosynthesis.

Figure 1

Schematic depiction of fluorescent probes. Oligodeoxynucleotide probes that anneal to several small RNAs are used in the experiments shown in Fig. 2. The positions at which the fluorescent probes bind to their target RNAs are shown relative to the full-length primary transcripts. 5′-Fluorescein labels are denoted by asterisks, with internal CY3 labels denoted by daggers. The fluorescent antisense RNA probes used to detect U14 snoRNA have been described previously (Samarsky et al. 1998).

Figure 2

Nuclear tRNA precursors are found predominantly in the nucleolus of S. cerevisiae. In situ hybridization of fluorescently labeled oligonucleotide probes to endogenous RNAs was carried out as described in the text. The left panel of each triplet shows staining with the probe specific for the RNA named at the left of the panels. The center panels show staining of the same cells as at left but with a probe directed against U14 snoRNA (red) and DNA detection with DAPI (blue). Merging of the left and center panels is shown at right, with overlap between the green and red signals shown in yellow. Overlap between green and blue appears as blue–green. (a–c) Introns to pre-tRNA^Leu3 and pre-tRNA^Trp are probed simultaneously (a,c). (d–f) The intergenic spacer of pre-tRNA^Arg/tRNA^Asp dimeric transcripts is probed (d,f). (g–i) U6 snRNA is probed (g,i). (j–l) Mature tRNA^Leu3 is probed (j,l).

Figure 3

Fluorescent probes for wild-type RNase P RNA and an inserted sequence. The proposed secondary structure of the S. cerevisiae RNase P RNA (RPR1 RNA) subunit is shown, including the long-distance base-pairing that contributes to tertiary structure. The positions of the artificial 20-nucleotide insertion are italicized and indicated by a bracket. CY3-labeled complementary oligonucleotide probe positions to either the wild-type RPR1 sequence or the inserted sequence are indicated by lines along the sequence.

Figure 4

RNase P is located primarily in the nucleolus in S. cerevisiae. All panels are stained for nuclear DNA with DAPI (blue). Probes for RNase P RNA or an artificial insert in RNase P RNA (Fig. 3) are shown in green in the left panels. The center panels show probes of the same cells for U14 snoRNA (red). The right panels show the left and center panels merged, with overlap between the green and red signals in yellow. (No merged panel is given for the center set, as there is no RNA signal in the left panel.) (a–c) The mature domain of wild-type RNase P RNA is probed (RPR1 RNA, a,c). (d–e) The probe to an artificial insert in RPR1 RNA is applied to wild-type cells, in which, as expected, it gives no signal above background. (f–h) The artificial insert probe is used (f,h) with cells having the modified RPR1 allele containing inserted sequences complementary to the probe.