Title: Structural Studies of the Integrator Complex -- pre-UsnRNA 3'-end Processing Machinery
Description: The Integrator complex (INT) is a metazoan-specific group of proteins associated with RNA polymerase II (Pol II) that has important functions in the 3'-end processing of noncodng RNAs, including uridine-rich small nuclear RNA (UsnRNA) and enhancer RNA (eRNA). Recently, INT has also been reported to be involved in Pol II transcriptional regulation of protein-encoding genes. INT contains at least 14 subunits, but the function of each subunit is difficult to predicted, because most subunits lack identifiable domains and display little similarity with other proteins. The endonuclease activity of INT is carried out by its subunit 11 (IntS11), which belongs to the metallo--lactamase superfamily and is a paralog of CPSF-73, the endonuclease for pre-mRNA 3'-end processing. IntS11 forms a stable complex with INT subunit 9 (IntS9) through their C-terminal domains (CTDs). This dissertation describes the crystal structure of the IntS9-IntS11 CTD complex at 2.1-Å resolution and summaries the structure-based biochemical and functional studies.

The complex is composed of a continuous nine-stranded -sheet with four strands from IntS9 CTD and five from IntS11 CTD. Highly conserved residues are located in the interface between the two CTDs. The structural observations on the complex are confirmed by yeast two-hybrid assays and coimmunoprecipitation experiments. Functional studies demonstrate that the Int9-IntS11 interaction is crucial for proper INT function in snRNA 3'-end processing. The dissertation also presents the structural studies of a newly found mammalian mRNA deNADding enzyme, Nudt12. We determined the crystal structure of mouse Nudt12 in complex with the deNADding product AMP and three Mg2+ ions at 1.6-Å resolution. The structure provides exquisite insights into the molecular basis of the deNADding activity within the NAD pyrophosphate. Previous studies have reported that NAD-capped mRNAs in mammalian cells are hydrolyzed by the DXO deNADding enzyme. Together with biochemical and functional studies, we demonstrate that Nudt12 is a second mammalian deNADding enzyme structurally and mechanistically distinct from DXO and targets different RNAs.