rRNA maturation in yeast cells depleted of large ribosomal subunit proteins

Abstract

The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins). They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU) proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein-rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i) how individual r-proteins control the productive processing of the major 5' end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii) the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events.

Figure 1. Large ribosomal subunit rRNA maturation pathways in S. cerevisiae.

Four of the five rRNAs found in mature ribosomes are derived from transcripts made by RNA polymerase I and processed through a series of endo- and exonucleolytic reactions. In (A) External transcribed spacer regions (ETS1, ETS2), Internal transcribed spacer regions (ITS1, ITS2), the transcription start site (+1) and major rRNA processing sites of primary rDNA transcripts are indicated. In (B) major pathways of precursor rRNA processing are shown.

Figure 2. Relative accumulation of large ribosomal subunit pre-rRNAs in conditional r-protein gene mutants as indicated by Northern blotting analyses.

Yeast strains expressing the indicated r-protein genes under control of the galactose-dependent GAL1 promoter were shifted to glucose medium in A) for 2 hours (white bars), 4 hours (light grey bars) or 8 hours (dark grey bar) and in B) and C) for 2 hours (light grey bars) or 4 hours (dark grey bars). For each strain the relative accumulation of 18S rRNA over 25S rRNA (A), 27SA2 pre-rRNA over total 27S pre-rRNA (B) and 7S pre-rRNA over total 27S pre-rRNA (C) in restrictive (glucose medium) versus permissive (galactose medium) conditions was determined by Northern blotting as described in Materials and methods. These relative accumulations are indicated in the y-axis of the diagrams in A) to C). Strains are placed in groups according to rRNA processing phenotypes.

Figure 3. Accumulation of 27SA₂- , 27SA₃- , 27SB_1L- and 27SB_1S pre-rRNA in conditional r-protein gene mutants as indicated by primer extension analyses.

Yeast strains expressing the indicated r-protein genes under control of the galactose-dependent GAL1 promoter were shifted for the specified times to glucose-containing medium. Primer extension analyses of LSU pre-rRNA was performed as described in Materials and methods. The diagrams in the lower panel are quantitative representations of the radioactive signals seen in the upper panel.

Figure 4. Accumulation of 3′ extended forms of 5.8S rRNA in conditional r-protein gene mutants as indicated by Northern blotting analyses.

Yeast strains expressing the indicated r-protein genes under control of the galactose dependent GAL1 promoter were shifted for the specified times to glucose containing medium. Detection of 3′ extended forms of 5.8S rRNA by northern blotting was performed as described in Materials and methods. Shown is analysis of wildtype yeast (BY4741) and of yeast strains in which separation of 5.8S rRNA precursors and 25S rRNA precursors through endonucleolytic cleavage in the ITS2 region of pre-rRNA was readily detectable.

Figure 5. Neo-synthesis and intracellular transport of tRNA and rRNA in conditional r-protein gene mutants as indicated by metabolic RNA labelling and nucleo-cytoplasmic fractionation.

Yeast strains expressing the indicated r-protein genes under control of the galactose-dependent GAL1 promoter were shifted for the specified times to glucose-containing medium. Pulse-labelling of total RNA (15 minutes) followed by nucleo-cytoplasmic fractionation and visualisation of newly synthesised RNA contained in nuclear (N) and cytoplasmic (C) fractions was performed as described in Materials and methods. Two point five times more RNA of nuclear than of cytoplasmic fractions was analysed.

Figure 6. rRNA – r-protein interactions as indicated by atomic resolution structure models of the eukaryotic mature cytoplasmic LSU.

PDB file 2ZKR from , based on a 8.7 A electron cryomicroscopy map of the mammalian ribosome in which rRNA and homology models of r-proteins were docked, was used in (A) – (D). The phosphate backbone of (parts of) LSU rRNA (in yellow, if not stated otherwise) and ribbon representations of 20 r-proteins with currently known 3D-localisation are shown (dark green). In (A) domain I and the 5′ region of domain II of LSU rRNA are visualised with the 5.8S rRNA in blue and a part of domain II forming a helical structure with the 5′ end of 5.8S rRNA in red. In (B) – (D) the LSU is seen in the crown view with the L1 stalk on the left, the (L7/L12-)Phospho-stalk on the right and the 5S rRNA in light green. In (B) - (D) 5.8S rRNA is highlighted in blue, its 3′ end in red and the 5′ end of 25S rRNA in violet. In (C) rRNA is shown transparent, with LSU rRNA domain V in violet and LSU rRNA domain II in red.