Abstract
The synthesis of eukaryotic ribosomes is a complex, energetically demanding process requiring the aid of numerous non-ribosomal factors, such as the PeBoW complex. The mammalian PeBoW complex, composed of Pes1, Bop1, and WDR12, is essential for the processing of the 32S preribosomal RNA. Previous work in Saccharomyces cerevisiae has shown that release of the homologous proteins in this complex (Nop7, Erb1, and Ytm1, respectively) from preribosomal particles requires Rea1 (midasin or MDN1 in humans), a large dynein-like protein. Midasin contains a C-terminal metal ion-dependent adhesion site (MIDAS) domain that interacts with the N-terminal ubiquitin-like (UBL) domain of Ytm1/WDR12 as well as the UBL domain of Rsa4/Nle1 in a later step in the ribosome maturation pathway. Here we present the crystal structure of the UBL domain of the WDR12 homologue from S. cerevisiae at 1.7 Å resolution and demonstrate that human midasin binds to WDR12 as well as Nle1 through their respective UBL domains. Midasin contains a well conserved extension region upstream of the MIDAS domain required for binding WDR12 and Nle1, and the interaction is dependent upon metal ion coordination because removal of the metal or mutation of residues that coordinate the metal ion diminishes the interaction. Mammalian WDR12 displays prominent nucleolar localization that is dependent upon active ribosomal RNA transcription. Based upon these results, we propose that release of the PeBoW complex and subsequent release of Nle1 by midasin is a well conserved step in the ribosome maturation pathway in both yeast and mammalian cells.
Highlights
The synthesis of eukaryotic ribosomes is a complex, energetically demanding process requiring the aid of numerous nonribosomal factors, such as the PeBoW complex
We demonstrate that nucleolar localization of WDR12 in U2OS cells is dependent upon active ribosomal RNA (rRNA) transcription and that midasin primarily localizes to the nucleoplasm
Structure of the UBL Domain of S. cerevisiae Ytm1—To further our structural knowledge of Ytm1/WDR12 (Fig. 1A), we determined a crystal structure of the UBL domain of Ytm1 from S. cerevisiae at 1.7 Å resolution (Fig. 1B)
Summary
Reagents—Normal rabbit IgG antibodies against midasin and WDR12 for use in immunohistochemistry were obtained from Sigma. Membranes requiring a secondary antibody for visualization were incubated with either goat anti-mouse IgG (1:10,000 dilution)- or goat anti-rabbit IgG (1:80,000 dilution)-conjugated HRP antibodies in 5% (w/v) nonfat milk and 1% (w/v) bovine serum albumin in TBST and incubated for at least 1 h at room temperature. Subsequent to this incubation, membranes were again vigorously washed three times with TBST. The Alexa Fluor 633 conjugate fluorochrome was imaged at room temperature on the Zeiss AxioCam MRm camera with Zen Pro 2012 (Blue Edition) software and processed with ImageJ
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