Synthesis of functional eukaryotic ribosomal RNAs in trans: development of a novel in vivo rDNA system for dissecting ribosome biogenesis - PubMed (original) (raw)

Synthesis of functional eukaryotic ribosomal RNAs in trans: development of a novel in vivo rDNA system for dissecting ribosome biogenesis

W Q Liang et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Active 18S and 25S ribosomal RNAs were produced in trans in yeast, from plasmids containing RNA polymerase II transcription signals and rDNA fragments with unique hybridization tags. Analyses were carried out in cells with temperature-sensitive RNA polymerase I. Functional rRNAs were derived from separate 18S and 5.8/25S rRNA coding units, however, active 25S rRNA could be produced only by cotranscription with 5.8S rRNA. The results demonstrate that the polycistronic organization of the large rDNA operon is not required for successful processing of rRNA or assembly of functional ribosomes. The split operon system should facilitate future efforts to dissect eukaryotic ribosome biogenesis.

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Figures

Figure 1

Figure 1

The large rDNA operon of S. cerevisiae can be successfully expressed in two parts. (A) Expression of small and large subunit rRNAs in trans. The yeast rDNA operon was divided at the sites indicated, and each segment was expressed from a different plasmid, under control of GAL7 promoters and terminators (closed and open boxes). The experimental 18S and 25S RNA coding units contained unique hybridization tags (○| and □|) and the plasmids harboring the 5′ and 3′ portions of the operon carried URA3 and TRP1 genes (see Materials and Methods). rDNA function was evaluated in yeast strain NOY504, which contains a ts RNA polymerase I mutation and mutations in URA3 and TRP1 (7). Plasmid pWL210 is identical to pWL184 except that it contains a 6-nt substitution in the 5′ ETS region (open rectangle) shown to base-pair with U3 snoRNA (472AAAGAG477→UCUUCA; ref. 8). Sites of pre-rRNA processing (A0, A1, etc.) are indicated by arrows. Sizes of the ETS and ITS segments are shown above the wild-type operon. E and F refer to sites used to prime PCR amplification. Growth and RNA levels are indicated at the right. The numbers above the experimental constructs refer to the nucleotide positions at which they were divided. Note that the dissections are imperfect, with at least a few nucleotides absent in each case (see also Fig. 2). (B) Phenotypic properties of the split operons. Transformants containing the plasmid pairs shown in A were streaked on galactose plates at 37°C and incubated for 7 days. The positive control contained a pair of plasmids with intact rDNA operons (pWL109 and pWL155), and the negative control contained vectors without rDNA inserts. Growth phenotypes are summarized in A. +, good growth (colonies evident in 7 days); −, no growth. (C) Northern hybridization analysis of rRNA expressed in trans. Cells were first grown in glucose medium at 25°C and then diluted into galactose medium. RNA was extracted after incubation at 37°C for 7 h (9). Northern assays were carried out using probes specific for unique hybridization tags in 18S and 25S RNAs. Plasmids pWL109 and pWL155 were used as a positive control. The levels of 18S and 25S RNAs are tabulated in A; + indicates wild-type level, and − indicates an undetectable level.

Figure 2

Figure 2

Secondary structure of the yeast ITS1 region. The structure is divided into five domains labeled I-V (adapted from ref. 30). Known processing sites A2, A3, B1L, and B1S are indicated by arrows. Site D corresponds to the 3′ end of mature 18S RNA. B1L and B1S cleavages give rise to long and short forms of 5.8S RNA. The sites used to divide the operon (•|), and the corresponding plasmids are identified. Bold nucleotides are known to be conserved among several yeast species (25).

Figure 3

Figure 3

The ITS1 region is required for production of 25S RNA. (A) Structure of ITS1 deletion variants. The strategy used to construct these plasmids is described in Fig. 1_A_, along with details for construction of plasmids pWL180 and pWL184 (see Materials and Methods). Novel 3′-rDNA plasmids pWL208, -203, and -204 were prepared by subcloning appropriate PCR fragments into pWL155 after double digestion with Xho_I and Mlu_I. All rDNAs were expressed with GAL7 promoters and terminators. (B) Growth properties of cells containing the plasmid pairs shown in A. Phenotypes were analyzed on galactose medium, as described in Fig. 1_B and are summarized in A. ± indicates extremely slow growth. (C) Northern hybridization analysis of rRNA expressed from the different split operons; details of the assay are provided in Fig. 1_C. Levels of 18S and 25S RNA are tabulated in A.

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