Protein translation by the ribosome is essential for cellular life but not much is known about the molecular mechanisms governing how ribosomes are made. Ribosome biogenesis is a major consumer of cellular energy and an incredibly complex pathway involving more than 300 trans-acting factors that is regulated by well-known tumor suppressors like p53 as well as protoncogenes such as cMyc and TOR, and is emerging in the field as a new target for cancer therapy. Currently we are taking a structure/function approach to decipher the roles of several protein complexes that are essential for ribosome assembly and cell viability. The mammalian PeBoW complex, which is composed of Pes1, Bop1, and WDR12, is essential for the processing of the 32S pre-ribosomal RNA. Although it has been well established that the PeBoW proteins play an essential role in ribosome processing, it is not well known how this complex forms, interacts with the maturing rRNA or gets released from pre-ribosomal particles in the nucleolus. It has been shown that in S. cerevisiae that each of the homologous proteins in this complex (Nop7, Erb1, and Ytm1, respectively) are members of the 27S A3 factors and that Rea1, a large dynein like protein is required for release of the complex from pre-ribosome particles. Rea1 (Midasin or MDN1 in humans) is the largest protein in S. cerevisiae; consisting of six concatenated ATPase domains, a 260kDa linker domain, and a C-terminal metal-ion dependent adhesion site (MIDAS) domain. Previous work has identified this Rea1 MIDAS domain as interacting with the N-terminal ubiquitin like (UBL) domain of Ytm1/WDR12 as well as Rsa4, another assembly factor in a subsequent step in pre-ribosome processing. One of our initial goals with this project was to understand how Midasin recognizes its substrates, WDR12 and Nle1 and removes them from pre-60S particles. We solved the crystal structure of the ubiquitin-like (UBL) domain of the WDR12 homolog from S. cerevisiae, which revealed that the domain contains a beta-grasp fold and a well conserved hydrophobic core. Through pull-down assays in Hek293 cells we were able to demonstrate that Midasin contains a C-terminal metal ion-dependent adhesion site (MIDAS) domain that specifically interacts with the N-terminal UBL domain of WDR12. Subsequent pull-downs revealed that the MIDAS domain of Midasin also binds to the UBL domain of Nle1, at a later step of the ribosome maturation pathway. The interaction between the MIDAS and UBL domains is dependent upon metal ion-coordination as removal of the metal or mutation of residues such as E78 of the WDR12 that is thought to coordinate the metal ion diminishes the interaction. We also identified that Midasin contains a well-conserved extension region upstream of the MIDAS domain that is uniquely required for binding WDR12 and Nle1. Mammalian WDR12 displays prominent nucleolar localization that is dependent upon active rRNA transcription and while Midasin displays prominent nucleoplasmic localization suggesting that the interactions between Midasin and WDR12 are transient. Take together our data suggest that the release of the PeBoW complex and subsequent release of Nle1 by Midasin is a well-conserved step in the ribosome maturation pathway and that Midasin recognizes its substrates, WDR12 and Nle1, through metal ion-coordination with a conserved glutamate residue and a conserved extension domain.
