Assembly of 5S ribosomal RNA is required at a specific step of the pre-rRNA processing pathway

Abstract

A collection of yeast strains surviving with mutant 5S RNA has been constructed. The mutant strains presented alterations of the nucleolar structure, with less granular component, and a delocalization of the 25S rRNA throughout the nucleoplasm. The 5S RNA mutations affected helix I and resulted in decreased amounts of stable 5S RNA and of the ribosomal 60S subunits. The shortage of 60S subunits was due to a specific defect in the processing of the 27SB precursor RNA that gives rise to the mature 25S and 5.8S rRNA. The processing rate of the 27SB pre-rRNA was specifically delayed, whereas the 27SA and 20S pre-rRNA were processed at a normal rate. The defect was partially corrected by increasing the amount of mutant 5S RNA. We propose that the 5S RNA is recruited by the pre-60S particle and that its recruitment is necessary for the efficient processing of the 27SB RNA precursor. Such a mechanism could ensure that all newly formed mature 60S subunits contain stoichiometric amounts of the three rRNA components.

Figure 1

Analysis of the small stable RNA in 5S RNA mutants. Wild-type and mutant strains were grown in YPD medium to an OD_600nm of 0.5 at 30°C. Total RNA was extracted, 5 μg were analyzed on an 8% polyacrylamide/8 M urea gel, and visualized by ethidium bromide staining. 5.8S rRNA, 5S RNA (mutant and wild-type), and tRNA are indicated by arrows or brackets. The picture corresponds to a negative exposure.

Figure 2

Morphology defects in 5S RNA mutants. Single cells of wild-type (WT), YOK69, YSC14, and YOK74 strains were isolated on YPD medium and incubated at 30°C for 1 d. Developing clones were observed using a Zeiss microscope with a 10× objective lens and Nomarski DIC optics.

Figure 3

Overexpression of RPL5 or LHP1 improves the growth of 5S RNA mutants. Dilutions of cultures from wild-type and mutant strains transformed with the RLP5 (A) or the LHP1 gene (B) were spotted on −Ura plates and incubated at 30°C or 37°C. (A) WT, YOK77, YOK69, and YOK71 strains were incubated at 30°C for 2 d or at 37°C for 3 d. YOK73, YOK72, YOK76, YSC14, and YOK74 strains were incubated at 30°C or 37°C for 6 d. (B) All strains were incubated for 2 d at 30°C. At 37°C, WT, YOK77, and YOK69 strains were incubated for 3 d, YOK71 strain for 4 d, and YOK 73, YOK72, YOK76, YSC14, and YOK74 strains for 6 d. Asterisks (*) indicate two strains containing the same mutated 5S rDNA but present at a different copy number.

Figure 4

Mutations in 5S RNA lead to a shortage in 60S ribosomal subunits and to the accumulation of halfmer polysomes. WT, YOK77, and YSC14 strains were grown in YPD medium to an OD_600nm 0.6–0.8 at 30°C. Cell extracts were resolved on 7–47% sucrose gradients. The gradients were analyzed by continuous monitoring at A_254nm. The peaks of free 40S and 60S ribosomal subunits and 80S ribosomes (free couples and monosomes) are indicated. Halfmer polysomes are indicated by arrows.

Figure 5

Alteration of the 27SB pre-rRNA processing in 5S RNA mutants. (A) The S. cerevisiae pre-rRNA processing pathway. The sequences corresponding to the mature 18S, 5.8S, and 25S rRNAs are represented as black bars, embedded in the 5′ and 3′ external transcribed spacers and in the ITS1 and ITS2. Processing of the primary transcript starts at site A0 in the 5′ external transcribed spacers. The resulting 33S pre-rRNA is processed at site A1 and A2 to give rise, successively, to the 32S pre-rRNA (not represented) and to the 20S and 27SA2 precursors. Cleavage at site A2 separates the pre-rRNA for the small and the large ribosomal subunits. The 20S precursor is cleaved at site D to yield mature 18S rRNA. Cleavage of 27SA2 pre-rRNA at site A3 is rapidly followed by digestion to site B1_S generating the 27SB_S precursor. The mature 25S rRNA and the 7S pre-rRNA are released from 27SB_S after cleavages at sites C1 and C2. The 7S pre-rRNA digestion generates the mature 3′ end of the 5.8S rRNA. For simplicity only the major processing pathway from 27SA2 pre-rRNA to the 5.8S_S and the 25S rRNA is shown, an alternative pathway generates the minor 5.8S_L rRNA, which is 7–9 nt longer at its 5′ end. (B) The processing of the 27SB pre-rRNA is slowed down in 5S mutants. WT, YOK77, and YSC14 strains were pulse-labeled for 1 min with methyl- [³H]methionine and chased for 1, 2.5, 5.5, 10, or 30 min with an excess of unlabeled methionine. Total RNA was extracted, denatured with glyoxal, run on a 1.2% agarose gel, transferred to a nylon membrane, and visualized by fluorography. The specific activities of the ³H-labeled RNA for WT, YOK77, and YSC14 were ∼7,000, 2,000,and 850 cpm/μg, respectively. 10,000 cpm were loaded on the first gel (WT and YOK77) and 8,000 cpm on the second gel (YSC14). Autoradiography was 10 times longer for the second gel than for the first one. The position of the pre-rRNAs and mature rRNA are indicated.

Figure 6

Overexpression of RPL5 or LHP1 in YOK74 improves the processing of the 27SB precursor. YOK74 mutant cells transformed with a vector sequence, the RPL5 gene, or the LHP1 gene in multicopy were pulse-labeled for 1 min with [methyl-³H]methionine and chased for 1, 2.5, 5.5, 10, and 30 min with an excess of unlabeled methionine. Total RNA was extracted, denatured with glyoxal, run on a 1.2% agarose gel, transferred to a nylon membrane, and visualized by fluorography. The specific activities of the ³H-labeled RNA for YOK74 + vector, + RPL5, or + LHP1 were ∼1,500, 12,000, or 12,200 cpm/ μg, respectively. Equal amounts of radioactivity were loaded on each gel corresponding to 22,000 cpm for YOK74 + vector, 140,000 cpm for YOK74, or 120,000 cpm for YOK74 + LHP1. Autoradiography was for 12 d for YOK74 + vector, 9 d for YOK74 + RPL5, or 7 d for YOK74 + LHP1. The positions of the pre-rRNAs and mature rRNAs are indicated.

Figure 7

The nucleolus of 5S RNA mutants is partially disorganized. (A) Electron micrograph after freeze electron substitution of the wild-type and YSC14 strains grown at 30°C. Substructures similar to the components of the nucleolus of higher eukaryotes are visualized: the fibrillar center (FC), the dense fibrillar component (DFC), and the granular component (GC). The fibrillar centers are not visible on the WT section. N, nucleoplasm. (B) Immunogold localization of the nucleolar protein Nop1 in the wild-type and YSC14 strains. Nop1 was detected using anti-Nop1 mAbs revealed with a secondary gold-conjugated antibody. Gold particles colocalized with the nucleolus in the two strains. N, nucleoplasm. Bars, 500 nm.

Figure 8

Nuclear delocalization of the 25S rRNA in 5S RNA mutants. The localization of the 35S pre-rRNA and the 25S mature rRNA was analyzed by in situ hybridization using digoxigenin-11dUTP-labeled probes specific for the 35S pre-rRNA (A) or the 25S rRNA (B) in the wild-type (WT), YOK69, and YSC14 strains. The probes were detected with antidigoxigenin antibodies gold conjugate. In A, as a control, the grids were pretreated with DNase-free RNase (YSC14 + RNase) before hybridization. N, nucleoplasm; Nu, nucleolus; NE, nuclear envelope; and P, cell wall. Bars, 500 nm.