Structural Basis Of Pre-mRNA Recognition By CFI_m And The Structure Of Yeast Pub1

The maturation of the 3'-ends of most mRNAs is catalyzed by multiple protein complexes, including the endo-nucleolytic cleavage of primary transcripts and addition of poly(A) tails to the upstream cleavage products. Nearly all the critical protein complexes involved in eukaryotic pre-mRNA 3'-end processing have been identified. Cleavage factor I (CFI_m), consisting of a 25 kDa subunit (CFI_m25) and one of the three larger subunits (CFI_m59, CFI_m68, CFI_m72), is required for the 3'-end cleavage. CFI_m binds to the UGUAA elements upstream of AAUAAA elements of the pre-mRNA substrates that facilitates pre-mRNA 3'-end processing complex assembly and enhance the rate and overall efficiency of poly(A) site cleavage in vitro. Sequence-specific binding of CFI_m to pre-mRNA directs A(A/U)UAAA-independent poly(A) addition through interacting with poly(A) polymerase and hFip1. When added to partially purified 3'-end processing factors, recombinant CFI_m25-CFI_m68 complex was sufficient to reconstitute poly(A) site cleavage activity in vitro. Repression of CFI_m25-CFI_m68 complex activity by knocking down CFI_m25 does not affect the HeLa cell viability, but increases upstream poly(A) site usage, suggesting CFI_m25 plays an important role in poly(A) site selection. Therefore, it is important to exploring the structure-function relationship of CFI_m. This thesis presents the structural basis for pre-mRNA recognition by CFI_m.CFI_m68 interacts with CFI_m25 through its N-terminal RRM domain (CFI_m68RRM). We determined the crystal structure of CFI_m25-CFI_m68RRM complex, revealing that CFI_m68RRM interacts with CFI_m25 through a novel RRM-protein interaction mode to form a tetramer. Mutagenesis analysis and pull-down experiment showed that CFI_m25 dimerization is crucial for CFI_m complex assembly, suggesting CFI_m complex is possibly a tetramer in vivo. We also determined the crystal structure of CFI_m25-CFI_m68RRM-RNA complex. The CFI_m25-CFI_m68RRM tetramer binds two UGUAA elements in the positively charged cavities of the CFI_m25 dimer via hydrogen-bonds, hydrophobic contacts and base pair stacking. The kinetic analysis demonstrates that CFI_m complex assembly increases pre-mRNA binding affinity, and the subsequent mutagenesis analysis reveals the RNA binding surface of CFI_m68, suggesting CFI_m68 may bind the immediately flanking region at 5'-end of the UGUAA element.This thesis also presents the structural investigation of yeast poly(U) binding protein (Pub1). Yeast poly(U)-binding protein (Pub1) is a major nuclear and cytoplasmic protein, containing three RNA recognition motif (RRM) domains (termed Pub1-RRM1, Pub1-RRM2 and Pub1-RRM3), which has been implicated as a regulator of cellular mRNA decay. Nearly 10% of all yeast mRNAs decay occurs in a Pub1-dependent manner. Pub1 binds to and stabilizes AU-rich element (ARE) and ARE-like sequence-containing transcripts by protecting them from degradation through the deadenylation-dependent pathway, and also binds to and stabilizes stabilizer element (STE)-containing transcripts by preventing their degradation via the nonsense-mediated decay (NMD) pathway. We determined the crystal structures of Pub1-RRM2 and the first two tandem RRM domains (Pub1-RRM12). Pub1-RRM1 and Pub1-RRM2 adopt the canonicalαβsandwich structures of RRM domains. Pub1-RRM12 forms a CV-N type domain-swapped dimmer by crystal packing. Size exclusion chromatography assay and analysitcal ultracentrifugation (AUC) showed Pub1-RRM12 is a monomer in solution. Mutagenesis analysis revealed five residues, located on the twoβ-sheets of Pub1-RRM1 and Pub1-RRM2, are critical for poly(U) binding. Kinetic analysis showed that all the three individual RRM domains can bind to a 10- or 15-base poly(U) segment with similar affinities, whereas Pub1-RRM12 binds to the 15-base poly(U) segment with the affinity approximately an order of magnitude higher than the 10-base poly(U) segment. Our studies provide structural and biochemical information for Pub1.