Roles of Dim2 in ribosome assembly

Abstract

In eukaryotes, ribosome assembly requires hundreds of conserved essential proteins not present in the mature particle. Despite their importance, the function of most factors remains unknown. This is because protein deletion often affects the composition of the entire particle. Additionally, many proteins are present in assembling ribosomes for extended times, which makes it difficult to pinpoint their role to a particular step. Here we have combined classical yeast biochemistry with experiments using recombinant proteins and RNA to study the role of Dim2 and its interaction with Nob1, the nuclease that generates the 3'-end of 18 S rRNA. Analysis of Dim2 mutants in which the interaction with Nob1 is disrupted demonstrates that this interaction between Dim2 and Nob1 is essential for optimal growth, and RNA binding experiments show that Dim2 increases Nob1 RNA affinity. Furthermore, our data indicate that Dim2 helps regulate Nob1 cleavage activity at the 3'-end of 18 S rRNA, as point mutants where this interaction is abolished in vitro accumulate pre-ribosomes containing Nob1 and 20 S rRNA in vivo. Interestingly, the site of interaction with Nob1 is mapped to the canonical RNA binding surface of a KH-like domain in Dim2, providing another example where an RNA-binding domain can be repurposed for protein interactions.

FIGURE 1.

Pre-18 S rRNA processing in yeast. A, schematic overview of the rRNA transcript and cleavage sites for 18 S production. The locations of the Northern probes used in Fig. 5 are shown with bars and letters under the RNA. B, order of early rRNA processing and transcription steps in yeast. For simplicity steps required for 60 S assembly are excluded.

FIGURE 2.

Dim2 interacts directly with Nob1. A, in vitro pulldowns with recombinant Nob1 and MBP-tagged Dim2. B, reverse in vitro pulldowns with recombinant MBP-Nob1 and Dim2. Control reactions show that Nob1 and Dim2 do not interact with MBP alone.

FIGURE 3.

Mutations in the Dim2 central KH-like domain abolish its interaction with Nob1. Nob1 interacts with MBP-tagged wild-type Dim2 but not the DDD/K, HR/E, or GXXG mutants. Asterisks denote Dim2 breakdown products, not Nob1, as confirmed by Western blotting (data not shown).

FIGURE 4.

Mutations in the Dim2 central KH-like domain are lethal. A yeast strain with Dim2 under a galactose-inducible promoter was transformed with pRS416TEF plasmids encoding wild type or Dim2 mutants and then grown on glucose- or galactose-containing plates.

FIGURE 5.

Northern analysis of pre-rRNA processing in strains containing Dim2 mutants. A, Nob1TAP; Dim2::Gal strains supplemented with plasmids encoding wild type or mutant Dim2 under the constitutive TEF promoter were grown in glucose-containing medium for 0, 8, or 12 h prior to harvest. Total RNA was extracted and separated on an agarose-formaldehyde gel. The gel was transferred onto a membrane and probed with the indicated oligonucleotides. The sequences and locations of these probes are listed in Table 1. B, accumulation of 20 S, 21 S, and 23 S pre-rRNAs after 8 h in glucose relative to the strain with plasmid-encoded wild type Dim2 (adjusted for loading differences by normalization to the U2 probe). These averages are obtained from three or more independent experiments.

FIGURE 6.

Nob1 binding to pre-ribosomes in strains containing Dim2 mutants. Nob1TAP; Dim2::Gal strains supplemented with plasmids encoding wild type or mutant Dim2 were grown in glucose-containing medium for 8 h prior to harvest. Lysates were then fractionated on 10–50% sucrose gradients. Proteins were TCA-precipitated, run on an SDS-PAGE gel, and probed for Nob1.

FIGURE 7.

Binding of Dim2ΔC strengthens the Nob1 RNA binding affinity. Data were fit to the Hill equation and yield K_1/2 values of 0.14 μm² and 0.26 μm⁴ in the presence and absence of Dim2ΔC, respectively. Different Hill coefficients were used because independent experiments indicate that the addition of Dim2 changes the stoichiometry of the Nob1·rRNA interaction from 4:1 to 2:1 (A. C. Lamanna and K. Karbstein, unpublished data). The different Hill coefficients preclude direct comparison of K_1/2 values. However, the different Hill coefficients do not change the finding that Dim2 increases Nob1 RNA binding affinity, as all data points in the presence of Dim2 are to the left of the corresponding ones in the absence.