Immature large ribosomal subunits containing the 7S pre-rRNA can engage in translation in Saccharomyces cerevisiae

Abstract

Evolution has provided eukaryotes with mechanisms that impede immature and/or aberrant ribosomes to engage in translation. These mechanisms basically either prevent the nucleo-cytoplasmic export of these particles or, once in the cytoplasm, the release of associated assembly factors, which interfere with the binding of translation initiation factors and/or the ribosomal subunit joining. We have previously shown that aberrant yeast 40S ribosomal subunits containing the 20S pre-rRNA can engage in translation. In this study, we describe that cells harbouring the dob1-1 allele, encoding a mutated version of the exosome-assisting RNA helicase Mtr4, accumulate otherwise nuclear pre-60S ribosomal particles containing the 7S pre-rRNA in the cytoplasm. Polysome fractionation analyses revealed that these particles are competent for translation and do not induce elongation stalls. This phenomenon is rather specific since most mutations in other exosome components or co-factors, impairing the 3' end processing of the mature 5.8S rRNA, accumulate 7S pre-rRNAs in the nucleus. In addition, we confirm that pre-60S ribosomal particles containing either 5.8S + 30 or 5.8S + 5 pre-rRNAs also engage in translation elongation. We propose that 7S pre-rRNA processing is not strictly required for pre-60S r-particle export and that, upon arrival in the cytoplasm, there is no specific mechanism to prevent translation by premature pre-60S r-particles containing 3' extended forms of mature 5.8S rRNA.

Keywords: DAPI, 4,6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; Mtr4/Dob1; NRD, Non-functional rRNA decay; RNA exosome; RNA helicase; Ribosome biogenesis; TRAMP complexes, Trf/Air/Mtr4 complexes; Translation; pre-rRNA processing; pre-rRNA, precursor rRNA; r-particles, ribosomal particles; r-proteins, ribosomal proteins; r-subunits, ribosomal subunits; yeast.

Figure 1.

Pre-60S r-particles containing 7S pre-rRNAs are exported to the cytoplasm in the dob1–1 mutant. Isogenic wild-type (MTR4) and dob1–1 (mtr4) strains were grown at 30°C in YPD medium to mid-log phase. Immunoprecipitation experiments were carried out using IgG-Sepharose and whole-cell extracts from wild-type (A) and dob1–1 (B) control cells (no TAP bait) or cells expressing TAP-tagged Nop7, Arx1, Lsg1, and L24A. RNA was extracted from the beads (lanes IP) or from an amount of total extract corresponding to 1/100 of that used for the immunoprecipitations (lanes T), separated on a 7% polyacrylamide-8M urea gel, transferred to a nylon membrane and subjected to northern hybridization with the 3 probes indicated in parentheses to detect 7S pre-rRNAs and mature 5.8S and 5S rRNAs. Probes are described in Figure 2 and Supplemental Table 2. Signal intensities were measured by phosphorimager scanning; values (below each IP lane) refer to the percentage of each RNA recovered after purification.

Figure 2.

Detection of pre-ribosomal particles by fluorescence in situ hybridization (FISH). (A) Primary structure of the 2 internal transcribed spacers, ITS1 and ITS2, of the 35S pre-rRNA. Processing sites are shown. The location of the 3 Cy-3 labeled probes used for FISH (purple) and of the oligonucleotides probes used for northern hybridization (black) is indicated. (B and C) Wild-type (MTR4) and dob1–1 (mtr4) cells were grown at 30°C in YPD medium to mid-log phase. Distinct pre-rRNAs were detected by FISH using Cy-3 labeled ITS1, ITS2–1 or ITS2–2 probes (red; purple in merge panels). Cells were further stained with DAPI to visualize the nucleoplasm (blue). All images shown were captured using identical exposure times and processed in the same manner.

Figure 3.

Pre-60S r-particles containing 7S pre-rRNAs engage in translation in the dob1–1 mutant. Wild-type (MTR4) and dob1–1 (mtr4) cells were grown at 30°C in YPD medium to mid-log phase. (A) Cells extracts were prepared and 10 A₂₆₀ units of each extract were resolved in 7–50% sucrose gradients and fractionated. RNA was extracted from each fraction and analyzed by northern blotting using probes f, e and 5S, which reveal 7S pre-rRNAs and mature 5.8S and 5S rRNAs, respectively. The position of free 40S and 60S r-subunits, 80S ribosomes and polysomes, obtained from the recorded A₂₅₄ profiles, are shown. (B) Signal intensities of the 7S pre-rRNAs (black dot, continuous line) and 5.8S_S rRNA (white squares, dashed line) were determined for each fraction by phosphorimager scanning and represented as arbitrary units.

Figure 4.

Pre-rRNA processing of the 7S pre-rRNAs in different mutants affected in 5.8S rRNA maturation. A wild-type strain and the indicated mutants were grown at 30°C in YPD medium to mid-log phase or shifted for 4 h to 37°C. Total RNA was prepared and 5 μg was separated on a 7% polyacrylamide-8M urea gel, transferred to a nylon membrane and subjected to northern hybridization with the 3 probes indicated in parentheses to detect 7S pre-rRNAs and mature 5.8S and 5S rRNAs. Note that probe e also reveals 5.8S + 30 and 5.8S + 5 pre-rRNAs in the rrp6Δ and rrp47Δ, and ngl2Δ mutants, respectively.

Figure 5.

Ability of pre-60S r-particles containing 3′ end extended precursors of 5.8S rRNAs to engage in translation in different mutant strains. A wild-type strain and the indicated mutants were grown at 30°C in YPD medium to mid-log phase. The mtr3–1 and rrp4–1 mutants were also shifted for 4 h to 37°C. Cell extracts were prepared and 10 A₂₆₀ units of each extract were resolved in 7–50% sucrose gradients and fractionated. RNA was extracted from each fraction and analyzed by northern blotting using probes f and e, which reveal 7S pre-rRNAs and mature 5.8S rRNAs, respectively. Note that probe e also reveals 5.8S + 30 and 5.8S + 5 pre-rRNAs in the rrp6Δ and rrp47Δ, and ngl2Δ mutants, respectively. The position of free 40S and 60S r-subunits, 80S ribosomes and polysomes, obtained from the recorded A₂₅₄ profiles, are indicated.

Figure 6.

Pre-60S r-particles containing 7S pre-rRNAs are exported to the cytoplasm in the rrp4–1 mutant at the non-permissive temperature. The rrp4–1 strain was grown at 30°C in YPD medium to mid-log phase (A) or shifted for 4 h to 37°C (B). Immunoprecipitation experiments were carried out using IgG-Sepharose and whole-cell extracts from untagged or TAP-tagged Nop7 and Lsg1 cells. RNA was extracted from the beads (lanes IP) or from an amount of total extract corresponding to 1/100 of that used for the immunoprecipitations (lanes T), separated on a 7% polyacrylamide-8M urea gel, transferred to a nylon membrane and subjected to northern hybridization with the probes indicated in parentheses to detect 7S pre-rRNAs and mature 5.8S and 5S rRNAs. Signal intensities were measured by phoshorimager scanning; values (below each IP lane) refer to the percentage of each RNA recovered after purification.