The Crystal Structure of the Ubiquitin-like Domain of Ribosome Assembly Factor Ytm1 and Characterization of Its Interaction with the AAA-ATPase Midasin

Abstract

The synthesis of eukaryotic ribosomes is a complex, energetically demanding process requiring the aid of numerous nonribosomal 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.Ribosomes are large macromolecular machines composed of four pieces of ribosomal RNA (rRNA) 2 and 80 (79 in yeast) associated ribosomal proteins (1, 2). Ribosomes are responsible for carrying out the synthesis of all proteins within a cell, and the eukaryotic ribosome is composed of two subunits known as the small subunit (40S) and the large subunit (60S). The assembly of ribosomes begins in the nucleolus with the transcription of the rRNAs. Three of the rRNAs are transcribed as a single polycistronic precursor (18S, 5.8S, and 25S), which must then be modified, folded, processed, and exported to the cytoplasm in a carefully orchestrated manner (3, 4). Ribosome biogenesis in eukaryotic cells is an incredibly complex and energetically demanding process that requires more than 200 essential nonribosomal assembly factors (3-6). Defects in the mammalian ribosome biogenesis pathway are linked to a group of human diseases that are collectively called ribosomopathies. These are all congenital, inherited disorders with a broad clinical spectrum that have perplexed researchers for years because they cause tissue-specific effects although ribosomes are essential in all cell types (7). Ribosome biogenesis has also been emerging as a new target for cancer therapy. Recent studies have shown that several important oncogenes, including cMYC, RAS, and PI3K, play key roles in promoting hyperactive ribosome biogenesis, and deregulated ribosomal DNA...

The AAA-ATPase MIDASIN 1 Functions in Ribosome Biogenesis and Is Essential for Embryo and Root Development

“…The substrates are targeted by Mdn1 through its C-terminal MIDAS-domain, which interacts with the N-terminal UBL domain of the substrates (Ulbrich et al, 2009;Bassler et al, 2010). The MIDAS-UBL interaction pattern is also observed in human cells (Romes et al, 2016). In this study, we determined that PES2 was physically associated with MDN1 through the MIDAS-UBL interaction pattern (Fig.…”

“…The yeast counterpart of MDN1 is localized in the nucleus and is required for nuclear export of the pre-60S ribosomal subunit (Galani et al, 2004). In human cells, the MDN1 counterpart can be detected in both the nucleus and cytoplasm (Romes et al, 2016) and also plays a role in 60S ribosome biogenesis (Raman et al, 2016). In Arabidopsis, the results of proteomic analyses have revealed the presence of MDN1 in both the nucleus and cytoplasm (Palm et al, 2016).…”

Structural Analysis Reveals Features of Ribosome Assembly Factor Nsa1/WDR74 Important for Localization and Interaction with Rix7/NVL2

“…Many steps of ribosome assembly take place within the nucleolus, including pre-rRNA transcription and early pre-rRNA processing and assembly steps (Thomson et al, 2013). Inhibition of Pol I pre-rRNA transcription has been shown to influence the localization of numerous ribosome assembly factors including NVL2 and WDR12 (Nagahama et al, 2004; Romes et al, 2016). Treatment of cells with low concentrations of actinomycin D, which inhibits Pol I transcription, led to a loss of the nucleolar localization of WDR74 and its redistribution to the nucleoplasm (Figure 4).…”

“…Rix7 is the earliest acting AAA-ATPase and appears to be involved in driving release of the essential assembly factor Nsa1 (Nop7 associated 1) from nucleolar Nop7-containing pre-60S particles (Kressler et al, 2008). The second AAA-ATPase, Rea1 (known as Midasin in higher eukaryotes), drives release of Erb1 and Ytm1 from early nucleolar pre-60S particles and Rsa4 from later nucleoplasmic pre-60S particles (Barrio-Garcia et al, 2016; Bassler et al, 2010; Romes et al, 2016; Thoms et al, 2016; Ulbrich et al, 2009). Rea1 contains a C-terminal MIDAS (metal ion-dependent adhesion site) that is necessary for recognizing the N-terminal Ubiquitin-Like (UBL) domains on both Ytm1 and Rsa4 (Bassler et al, 2010; Bassler J, 2014; Romes et al, 2016; Ulbrich et al, 2009).…”

NOP7 interacts with β-catenin and activates β-catenin/TCF signaling in hepatocellular carcinoma cells

“… 14 NOP7, BOP1, and WDR12 form the PeBoW complex and promote cell proliferation. 15 , 16 Moreover, NOP7 is essential for the proliferation and tumorigenicity of breast cancer cells. 17 However, its roles in HCC remain largely unknown.…”