JBC Papers in Press. Published on November 24, 2015 as Manuscript M115.693259 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M115.693259 Interactions between mammalian WDR12 and Midasin The Crystal Structure of the Ubiquitin-Like Domain of Ribosome Assembly Factor Ytm1 and Characterization of its Interaction with the AAA-ATPase Midasin Erin M. Romes, Mack Sobhany, and Robin E. Stanley* From the Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, 111 T. W. Alexander Drive, Research Triangle Park, North Carolina 27709, USA Running Title: Interactions between mammalian WDR12 and Midasin *To whom correspondence should be addressed: Robin E. Stanley, Ph.D, Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health Building 101, Room F378, 111 T. W. Alexander Drive, Research Triangle Park, NC 27709, Telephone: (919)541-0270; E-mail: [email protected] Downloaded from Keywords: ribosome assembly, rRNA processing, crystallography, ATPases associated with diverse cellular activities (AAA), metal ion‐protein interaction http://www.jbc.org/ ABSTRACT and the interaction is dependent upon metal The synthesis of eukaryotic ribosomes ion-coordination as removal of the metal or is a complex, energetically-demanding process mutation of residues that coordinate the metal requiring the aid of numerous non-ribosomal ion diminishes the interaction. Mammalian factors such as the PeBoW complex. The WDR12 displays prominent nucleolar by guest on April 26, 2017 mammalian PeBoW complex, comprised of localization that is dependent upon active Pes1, Bop1, and WDR12, is essential for the rRNA transcription. Based upon these results processing of the 32S pre-ribosomal RNA. we propose that release of the PeBoW Previous work in Saccharomyces cerevisiae complex and subsequent release of Nle1 by has shown that release of the homologous Midasin is a well-conserved step in the proteins in this complex (Nop7, Erb1, and ribosome maturation pathway in both yeast Ytm1, respectively) from pre-ribosomal and mammalian cells. particles requires Rea1 (Midasin or MDN1 in _____________________________________ humans), a large dynein-like protein. Midasin Ribosomes are large macromolecular contains a C-terminal metal ion-dependent machines comprised of four pieces of adhesion site (MIDAS) domain that interacts ribosomal RNA (rRNA) and 80 (79 in yeast) with the N-terminal ubiquitin-like (UBL) associated ribosomal proteins (1,2). domain of Ytm1/WDR12 as well as the UBL Ribosomes are responsible for carrying out the domain of Rsa4/Nle1 in a later step in the synthesis of all proteins within a cell, and the ribosome maturation pathway. Here we eukaryotic ribosome is composed of two present the crystal structure of the UBL subunits known as the small subunit (40S) and domain of the WDR12 homologue from S. the large subunit (60S). The assembly of cerevisiae at 1.7 Å resolution and demonstrate ribosomes begins in the nucleolus with the that human Midasin binds to WDR12 as well transcription of the rRNAs. Three of the as Nle1 through their respective UBL rRNAs are transcribed as a single poly- domains. Midasin contains a well-conserved cistronic precursor (18S, 5.8S, and 25S), extension region upstream of the MIDAS which must then be modified, folded, domain required for binding WDR12 and Nle1 processed and exported to the cytoplasm in a 1 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Interactions between mammalian WDR12 and Midasin carefully orchestrated manor (3,4). Ribosome in yeast, Nop7, Erb1, and Ytm1 are all biogenesis in eukaryotic cells is an incredibly essential proteins required for the processing complex and energetically demanding process of the 27SA3 pre-rRNA (17,18). All that requires more than 200 essential non- components of the PeBoW/Nop7 complex are ribosomal assembly factors (3-6). thought to be multifunctional proteins and Defects in the mammalian ribosome have been linked with roles in various cellular biogenesis pathway are linked to a group of processes including DNA replication, cell human diseases that are collectively called cycle regulation and cardiac function in ribosomopathies. These are all congenital, addition to ribosome biogenesis (19,20). The inherited disorders with a broad clinical PeBoW/Nop7 complex is thought to primarily spectrum that have perplexed researchers for localize to the nucleolus. The complex is not years because they cause tissue-specific found on ribosomal particles within the effects even though ribosomes are essential in nucleoplasm or cytoplasm, indicating that the all cell types (7). Ribosome biogenesis has PeBoW complex must be released from pre- also been emerging as a new target for cancer ribosomal particles prior to their transport out therapy. Recent studies have shown that of the nucleolus (16,18,21). several important oncogenes, including One way in which ribosome cMYC, RAS and PI3K, play key roles in maturation factors are released from pre- Downloaded from promoting hyperactive ribosome biogenesis, ribosome particles is through the aid of three and deregulated ribosomal DNA transcription different AAA-ATPases, Rea1/Midasin, Rix7, is a requirement for the transformed and Drg1/Afg2, which all utilize both phenotype (8). Moreover, studies have also nucleotide binding and hydrolysis to drive http://www.jbc.org/ shown that the nucleolus is a key element in release of distinct ribosome maturation factors regulating the cellular stress response and is (reviewed in (22)). Rea1 is responsible for directly involved in regulating the activity of driving release of both the Nop7 complex and the tumor-suppressor p53 in response to stress the maturation factor Rsa4 from pre-60S (9-13). Furthering our understanding of the particles in S. cerevisiae. Rea1 is the largest by guest on April 26, 2017 ribosome biogenesis pathway is essential for protein in S. cerevisiae and contains an N- the development of new cancer therapeutics terminal domain, six concatenated ATPase and treatments for ribosomopathies. domains, a 260kDa linker domain, and a C- The majority of what is known about terminal MIDAS domain that is well eukaryotic ribosome biogenesis is based on conserved across all eukaryotes (23). Electron extensive studies in the budding yeast S. microscopy studies reveal that Rea1 has a cerevisiae (recently reviewed in (4)). While large AAA motor domain connected to a the overall process is thought to be well flexible tail-like structure, which contains the conserved among eukaryotes, much less is MIDAS domain at the end (24). Electron known about ribosome biogenesis in higher microscopy studies also indicate that Rea1 organisms (14). One complex that has been contacts pre-ribosome particles adjacent to the well characterized in both yeast and Rix1-Ipi1-Ipi3 subcomplex with the tail being mammalian cells is the PeBoW complex able to reach the pre-ribosomal factor Rsa4 (Nop7 complex in S. cerevisiae). The PeBoW (24). Further studies in S. cerevisiae complex was named for the mammalian demonstrated that Rea1 and Rsa4 interact with assembly proteins Pes1 (Pescadillo), Bop1 one another in vivo and in vitro through the (block of proliferation), and WDR12 (WD MIDAS domain of Rea1 and the conserved N- repeat domain 12), while the yeast terminal UBL domain of Rsa4, which was homologues are Nop7, Erb1, and Ytm1 previously referred to as the MIDO (MIDAS respectively. Knockdown of any of the interacting) domain (24). Interaction between proteins within the mammalian PeBoW Rea1 and Rsa4 coupled with ATP hydrolysis complex blocks processing of the large triggers release of Rsa4 from pre-ribosomal subunit 32S pre-RNA and triggers p53- particles, likely through a large-scale dependent cell cycle arrest (15,16). Similarly mechanical conformational change (22,24). 2 Interactions between mammalian WDR12 and Midasin The mammalian homologue of Rsa4 is called structural information for WDR12, and Notchless (Nle1) and was so named because it suggest that interactions between Midasin with is a direct regulator of the Notch signaling the UBL domains of WDR12 and Nle1 are pathway in Drosophila melanogaster in important evolutionarily conserved addition to its role in ribosome maturation interactions in the ribosome assembly (25). pathway. In addition to driving release of Rsa4, Rea1 has also been shown to drive release of EXPERIMENTAL PROCEDURES the Nop7 complex from nucleolar pre- ribosome particles in S. cerevisiae. Reagents – Normal rabbit IgG Bioinformatic analysis revealed that the N- antibodies against Midasin and WDR12 for terminal UBL domain of Rsa4 is homologous use in immunohistochemistry were obtained to the N-terminal UBL domain of Ytm1, from Sigma Life Science. Normal mouse suggesting that Rea1 could interact with Ytm1 antibodies conjugated with horseradish through its MIDAS domain (21). Earlier work peroxidase against GST (B-14) and His (H3) in mice also suggested that the N-terminal for use in Western Blots were obtained from domain of WDR12 showed similarity to the Santa Cruz Biotechnology. Monoclonal N-terminal domain of Nle1, and it was mouse antibody against the Strep-Tag peptide Downloaded from originally referred to in the literature as the and normal rabbit antibody against Actin for Nle1 domain (26). Subsequent experiments in use as primary antibodies in Western Blots S. cerevisiae demonstrated that Ytm1 interacts