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. Ribosome assembly 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 including Nsa1/Rix7 and Grc3/Las1 that are essential for ribosome assembly and cell viability. Nsa1 (WDR74 in mammals) and Rix7 (NVL2 in mammals) are two assembly factors required for production of the large ribosomal subunit. Rix7 is a type-II double ring AAA-ATPase that shares significant homology with the well-studied CDC48/p97. Nsa1 binds to late-stage nucleolar pre-60S ribosome particles and its release from pre-60S particles is driven by Rix7. The precise role for Nsa1 in ribosome assembly remains unclear. Through multiple structural analyses we provide the first insight into the function of Nsa1. Nsa1 is comprised of an N-terminal 7-bladed beta propeller followed by a flexible C-terminal tail that extends away from the WD40 domain. The WD40 domain of Nsa1 has a well-conserved surface that plays an important role in the association of the protein with Rix7, while the C-terminal tail is crucial for the proper localization of the protein within the nucleolus. Las1 was recently discovered as the long-sought after endoribonuclease which cleaves at the C2 site within the internal transcribed spacer 2 (ITS2) during ribosome assembly. Cleavage at the C2 site is an essential step during ribosome assembly because it separates the precursors of the 5.8S and 25S rRNA, triggers the further processing of the 5 end of the 26S pre-rRNA, and primes pre-60S particles for transit from the nucleolus to the nucleoplasm. While it is now established that Las1 is the C2 endoribonuclease the molecular mechanisms governing C2 cleavage are unclear. Mutations in the mammalian Las1 gene have also been linked to neurological dysfunction, underscoring the need to further understand the activity of this enzyme. Through a series of in vivo and in vitro studies we determined that Las1 is dependent upon its binding partner, the polynucleotide kinase Grc3 to stimulate specific endonuclease activity. Together Grc3 and Las1 assemble into a tetrameric complex composed of a dimer of Grc3/Las1 heterodimers. We also discovered that there is functional cross-talk between the nuclease and kinase domains of Las1 and Grc3 allowing for an exquisite level of control of enzymatic activity. All together our data provide the basis for understanding the molecular details of specific cleavage at the C2 site by higher-order assembly of the well-conserved and essential Grc3-Las1 complex.
