Function of pICln in regulating PRMT5 activity through the combined interaction of the pICln-SmD3 complex

The methylosome is a 20 S protein complex that is thought to regulate snRNP biogenesis through interactions with pICln and PRMT5 mediated arginine methylation of the common snRNP Sm proteins (Friesen et al., 2001 b) A coexpressed complex containing PRMT5, pICln, and SmD3 was used to determine the specific role of pICln in t methylosome function. Beyond earlier studies that characterize pICln as a component of the methylosome, I have demonstrated that (1) pICln selectively activates PRMT5 for methylation of Sm proteins through direct allosteric interactions with the regulatory domain of PRMT5, (2) it plays an active role in the methylation and release of SmD3 from methylation with PRMT5, and (3) it contributes to the formation of a higher ordered PRMT5 complex displaying the highest levels of PRMT5 activity. Coexpression of mutant PRMT5 complexes establishes a functional role in the modular domains of pICln and shows that the release of a pICln-SmD3 product complex is dependent on methylation of SmD3. As a coactivator of PRMT5 methylation, pICln sequesters Sm proteins in a minimal monomeric state for subsequent snRNP assembly. In an extension to the known interaction between pICln and SmD3, I have defined a vital interaction between pICln and a conserved arginine in SmD3 by mutagenesis. As this arginine facilitates a vital non-specific interaction between Sm proteins, a mechanism of preventing Sm-Sm protein association and snRNP assembly is proposed. Further analysis of protein structure by sedimentation velocity and circular dichroism suggests that the conformation of pICln is an essential component for the high affinity interaction between pICln and SmD3. A rational model of the pICln-SmD3 complex is presented that supports protein-protein interaction studies and the hydrodynamic character of a pICln-SmD3 complex. The recombinant reconstitution of a PRMT5 complex sets a precedent for studying protein methylation in the context of large macromolecular assemblies. These studies define several fundamental roles for pICln in regulating snRNP assembly through protein-protein interactions with SmD3 and activating arginine methylation by PRMT5