Structural and biochemical studies of the Gemin6/7 heterodimer from the human SMN complex

The survival of motor neurons (SMN) protein, product of the disease gene of the common neurodegenerative disease spinal muscular atrophy (SMA), is part of a large multi-protein complex (SMN complex) that also contains Gemins2–7. The SMN complex functions as an assembly machine for small nuclear ribonucleoproteins (snRNPs), the major components of the spliceosome. How the SMN complex mediates the assembly of snRNPs, each of which contains a snRNA surrounded by a ring of seven Sm proteins, is not known. As part of an overall effort of the Van Duyne lab to study the structure and function of the SMN complex, the goal of this thesis work is to investigate the structures, biochemical and biophysical properties of individual proteins and stable subcomplexes in this system. Here I report the crystal structure of two components of human the human SMN complex, Gemin6 and Gemin7. The structure of the Gemin6/Gemin7 complex was determined by a combination of MAD and molecular replacement methods at a 2.0 Å resolution. Interestingly, although Gemin6 and Gemin7 have no significant sequence similarity with Sm proteins, both adopt a canonical Sm fold. Moreover, Gemin6 and Gemin7 exist as a heterodimer, and interact with each other via an interface similar to that which mediates interactions among the Sm proteins. GST pull down binding assays show that the Gemin6/Gemin7 complex binds to Sm proteins. Recombinant purified Sm proteins and Gemin6/7 complex were used successfully in the in vitro snRNP reconstitution assembly assays and provided a quick and straight forward method to examine snRNP core assembly in general. Significant progress was also made in the characterization of the SMN/Gemin2 complex although diffraction quality crystals were never obtained. Together, the structural, biochemical and biophysical findings of the Gemin6/7 complex provide a framework for considering how the SMN complex, with Gemin6 and Gemin7 as tools, organizes the Sm proteins for formation of the Sm ring on its snRNA targets