Identification of the binding site of Rlp7 on assembling 60S ribosomal subunits in Saccharomyces cerevisiae

Abstract

Eukaryotic ribosome assembly requires over 200 assembly factors that facilitate rRNA folding, ribosomal protein binding, and pre-rRNA processing. One such factor is Rlp7, an essential RNA binding protein required for consecutive pre-rRNA processing steps for assembly of yeast 60S ribosomal subunits: exonucleolytic processing of 27SA3 pre-rRNA to generate the 5' end of 5.8S rRNA and endonucleolytic cleavage of the 27SB pre-rRNA to initiate removal of internal transcribed spacer 2 (ITS2). To better understand the functions of Rlp7 in 27S pre-rRNA processing steps, we identified where it crosslinks to pre-rRNA. We found that Rlp7 binds at the junction of ITS2 and the ITS2-proximal stem, between the 3' end of 5.8S rRNA and the 5' end of 25S rRNA. Consistent with Rlp7 binding to this neighborhood during assembly, two-hybrid and affinity copurification assays showed that Rlp7 interacts with other assembly factors that bind to or near ITS2 and the proximal stem. We used in vivo RNA structure probing to demonstrate that the proximal stem forms prior to Rlp7 binding and that Rlp7 binding induces RNA conformational changes in ITS2 that may chaperone rRNA folding and regulate 27S pre-rRNA processing. Our findings contradict the hypothesis that Rlp7 functions as a placeholder for ribosomal protein L7, from which Rlp7 is thought to have evolved in yeast. The binding site of Rlp7 is within eukaryotic-specific RNA elements, which are not found in bacteria. Thus, we propose that Rlp7 coevolved with these RNA elements to facilitate eukaryotic-specific functions in ribosome assembly and pre-rRNA processing.

Keywords: RNA binding protein; Rlp7; crosslinking and analysis of cDNAs (CRAC); ribosomal protein L7; ribosome biogenesis.

FIGURE 1.

Yeast ribosome assembly. (A) Pre-rRNA processing pathway. Eukaryotic pre-rRNA processing occurs through a series of consecutive steps. The locations of the ETS and ITS sequences (lines), pre-rRNA processing sites (vertical ticks), and mature rRNA sequences (boxes) are shown. The solid arrow marks the 27SA₃ pre-rRNA processing step, which is blocked in the absence of Rlp7 and the other A₃ factors. The dashed arrows indicate the 27SB pre-rRNA processing step, which also is blocked in rlp7 mutants. The 40S subunit contains 18S rRNA and the 60S subunit contains 5.8S, 25S, and 5S rRNAs. 5S rRNA is transcribed and processed separately and is not shown. (B) Cartoon of the solvent-accessible interface of pre-60S ribosomes containing mature rRNA domains I–VI and ITS2 in the hairpin conformation. Domain IV is on the back in this view (subunit interface) and thus is not labeled. (C) Cartoon representation of base-pairing between 5.8S and 25S rRNAs in domain I. Sequences of mature rRNA are black, and ITS1 and ITS2 are gray. Boxes indicate regions where 5.8S rRNA base-pairs with 25S rRNA. Helix numbers are listed for 25S rRNA. Helix 10 is also known as the ITS2-proximal stem. Gray circles indicate the binding sites of Rlp7, Erb1, Nop12, Nop15, and Cic1 on preribosomes. The A₃, B_1S, and C₂ processing sites are indicated.

FIGURE 2.

Rlp7 crosslinks to ITS2 and the ITS2-proximal stem of domain I. (A) Purification of RNA crosslinked to Rlp7. An autoradiogram of affinity purified Rlp7 and crosslinked RNA is shown. His-tagged Rlp7 (∼38 kDa) with crosslinked RNA and linkers (∼21–26 kDa) is expected to migrate around 59–64 kDa in an SDS–polyacrylamide gel. Results from the untagged parent strain, BY4741, are shown as a negative control. A faint band ∼55 kDa is sometimes seen in the negative control, consistent with published observations (Granneman et al. 2009). (B) TapeStation-generated gel image of cDNA libraries. The expected length of cDNA libraries derived from 15- to 30-nucleotide RNA fragments is 130–145 bp. For reference the 25- and 1500-bp markers are shown in all lanes. The negative control consistently yielded no detectable cDNA. (C) Graphical view of Rlp7 CRAC sites on 5.8S and 25S rRNAs and ITS1, ITS2, and 3′ETS. The number of hits to each site is indicated on the y-axis, and the frequency is indicated above (percentage of time site was observed).

FIGURE 3.

Rlp7 interacts with a neighborhood of proteins that bind to or near ITS2 and the proximal stem. (A) Two-hybrid interaction map. Two-hybrid assays were carried out to test for interactions between the ITS2 neighborhood of proteins (Rlp7, Nop15, Cic1) and several other factors required for 27S pre-rRNA processing. Relative strengths of interactions are shown with dark gray, thick lines representing strong interactions and light gray, thin lines representing weak interactions as determined by growth of two-hybrid strains on media containing increasing concentrations (1, 2.5, 5, and 50 mM) of 3-aminotriazole (3-AT). (B) High-salt wash of Rlp7-associated proteins. Rlp7-HTP was affinity purified from yeast cell lysates, and complexes were washed with increasing concentrations of NaCl (150, 450, 1000 mM) to remove weakly associated proteins. One-step indicates that proteins were resolved after one-step purification on IgG-coated beads, and two-step indicates that proteins were resolved after two-step purification on IgG- and nickel-coated beads.

FIGURE 4.

The accessibility of nucleotides in ITS2 to DMS chemical modification is altered after Rlp7 depletion. (A) In vivo DMS probing of the GAL-RLP7 strain grown in galactose (Gal)-containing or glucose (Glu)-containing medium. Controls include no DMS control (−) and stop control (stop), in which β-mercaptoethanol was added to quench the reaction before addition of DMS. The corresponding sequencing ladders are shown (A, U, G, C). The 25S-ITS2 (lanes 1–8) and 3140-ITS2 (lanes 9–16) oligonucleotides were used for primer extension of extracted RNA. Nucleotides that become more modified in the absence of Rlp7 are indicated by closed blue circles, and nucleotides that became less modified are indicated by open blue circles. Nucleotide positions are designated for 35S pre-rRNA. The same RNA samples and loading were used for both sets of primer extensions. (B) Locations of nucleotides that are more (closed blue circles) or less (open blue circles) modified in the absence of Rlp7 are displayed on the predicted ring (Joseph et al. 1999) or hairpin (Yeh and Lee 1990) structure of ITS2. ITS2 contains nucleotides from sites E to C₁. Nucleotides that are modified in wild-type cells are circled in green. Orange lines represent the binding sites of Rlp7, Nop15, Cic1, and Nop12 determined by CRAC (Granneman et al. 2011). Crosslinked nucleotides are highlighted in yellow. On the hairpin structure, locations of primers used for primer extension are shown as dashed arrows (25S-ITS2, 3140-ITS2), and the location of the proximal stem is indicated. (C) PyMOL representation of the solvent accessible surface of the yeast 60S rRNA (PDB file 3U5H) (Ben-Shem et al. 2011). Domain I is blue (5.8S rRNA dark blue, 25S rRNA light blue), the 5′ end of 5.8S rRNA is green, and protein binding sites are shown in space-fill view. The binding site of Nop12 is red, and the binding sites of Erb1, Rlp7, and Nop7 are orange.