Yeast ribosomal protein L7 and its homologue Rlp7 are simultaneously present at distinct sites on pre-60S ribosomal particles

Abstract

Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7(L)b within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.

Figure 1.

Rlp7-associated pre-ribosomal particles contain the L7 r-protein. Wild-type and Rlp7-TAP cells were mixed in equal proportions prior to complex purification. The log₂ of SILAC ratios (median value of Wild-type/Rlp7-TAP peptide ratio) were plotted against the sum of the intensity of all the peptides for each protein. Dots are coloured according to protein function: pre-60S factors (red), 60S r-proteins (Rpl, blue), r-stalk proteins (P0/P1/P2, light blue), 40S r-proteins (Rps, green) and proteins of other different functions (grey). Yellow stars indicate the Rlp7 and L7 values. The identity of pre-60S factors specifically enriched (Group 1) or not (Group 2) is indicated below the graph. These factors are listed from their highest to lowest intensity values (see Supplementary Data Set S1).

Figure 2.

Rlp7 and L7 association with pre-ribosomal particles is not mutually exclusive. Extracts were prepared from cells co-expressing Rlp7-HA and L7B-TAP or, as a control, Rlp7-HA and untagged L7B. Total extracts (T) and L7B-TAP affinity-purified samples (IP) were analysed by western blotting. Co-precipitation of Rlp7-HA, Has1, Nop1 and r-proteins L1 and L35 was tested with specific antibodies. The asterisk corresponds to a L7B-TAP cross-reaction.

Figure 3.

Association of Rlp7 and L7 with r-particles. HTP-tagged Rlp7 and L7 were affinity-purified from extracts of the indicated strains. RNA was isolated from total extract (T) and purified samples (IP) and analysed by northern blotting. (A) Large pre- and mature rRNAs. (B) Small pre- and mature rRNAs. Probes (in parentheses) are described in Supplementary Figure S1. Signal intensity was measured by phosphorimager scanning; values (below each IP lane) refer to the percentage of each RNA recovered after purification.

Figure 4.

Identification of Rlp7 and L7 binding sites on pre- and mature rRNAs. The histograms, plotted using the Integrative Genomics Viewer software, display the sequences identified on CRAC analysis and the number of hits mapped to the rDNA. CRAC was performed with Rlp7-HTP (Rlp7) and L7B-HTP rpl7AΔ (L7B) cells. The maximum number of hits in the main peaks is shown.

Figure 5.

Localization of the CRAC interaction sites of Rlp7 with pre-rRNA sequences displayed on the ‘hairpin model’ (A) and on the ‘ring model’ (B) for yeast ITS2 secondary structure [for a reference, see (45)]. The CRAC sites are highlighted in yellow; blue circles indicate frequently mutated residues found in the experiments (see Supplementary Figure S8). The Nsa3 and Nop15 CRAC sites, as described in (7), are represented as purple and green, respectively. The location of the CRAC sites cleavage sites C₁, C₂ and E are also indicated.

Figure 6.

Localization of the CRAC interaction sites of L7 in the secondary structure of domain II of 25S rRNA (A) and 5S rRNA (B). The structures, residues and helix (H) numbers were taken from the Comparative RNA Web Site (46). The eukaryotic expansion segment ES7^L is labelled with a dash box. Red circles indicate rRNA residues situated in close proximity of L7 (closer than 5 Å) in the structure of yeast 60S r-subunit [PDB file 3U5H; (19)]. The CRAC sites are highlighted in yellow; blue circles indicate frequently mutated residues found in the experiments.