Most eukaryotic pre-mRNAs undergo 3′-end cleavage and polyadenylation prior to their export from the nucleus. A large number of proteins in several complexes participate in this 3′-end processing, including cleavage and polyadenylation specificity factor (CPSF) in mammals. The CPSF can be further divided into two sub-complexes: mPSF (mammalian polyadenylation specificity factor) which recognizes the AAUAAA polyadenylation signal (PAS) in the pre- mRNA, and mCF (mammalian cleavage factor) which cleaves the RNA. mPSF consists of CPSF160, CPSF30, WDR33, and hFip1. This thesis shows that AAUAAA PAS is recognized with ∼3 nM affinity by the CPSF160–WDR33–CPSF30 ternary complex, while the proteins alone or the binary complexes do not bind the PAS with high affinity. Furthermore, it is shown that mutations of residues in CPSF30 that have van der Waals interactions with the bases of the PAS lead to a sharp reduction in the affinity. Finally, variations of the AAUAAA or removing the bases downstream also reduce the binding significantly. This thesis goes on to characterize the structure of the CPSF30—hFip1 complex, which was not observed in the previous EM structures of the mPSF. It was known that CPSF30 ZF4–ZF5 recruits the hFip1 subunit of CPSF, although the details of this interaction have not been characterized. Here we report the crystal structure of human CPSF30 ZF4–ZF5 in complex with residues 161–200 of hFip1 at 1.9 Å. Unexpectedly, the structure reveals one hFip1 molecule binding to each ZF4 and ZF5, with a conserved mode of interaction. Mutagenesis studies confirm that the CPSF30–hFip1 complex has 1:2 stoichiometry in vitro. Mutation of each binding site in CPSF30 still allows one copy of hFip1 to bind, while mutation of both sites abrogates binding. Our fluorescence polarization binding assays show that ZF4 has higher affinity for hFip1, with a Kd of 1.8 nM. We also demonstrate that two copies of the catalytic module of poly(A) polymerase (PAP) are recruited by the CPSF30–hFip1 complex in vitro, and both hFip1 binding sites in CPSF30 can support polyadenylation.

The purpose of this experiment was to determine if nucleic acid sequence based amplification (NASBA) is a suitable application for the differentiation of body fluids that might comprise a forensic evidence sample. NASBA is a sensitive RNA transcription based amplification system. NASBA could theorhetically be used for bodily fluid identification based upon amplification of tissue-specific mRNA transcripts present in a given forensic sample.Amplification of both Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Matrix Metalloproteinase 1 1 (MMPmRNA transcripts were used to determine that NASBA could amplify body fluid transcripts and whether it could distinguish between menstrual and non-menstrual blood, respectively. GAPDH is a housekeeping gene that is constituently expressed and its mRNA transcripts could therefore be used to determine whether non-menstrual blood could be amplified using the NASBA procedure. MMP 11 is a menstrual cycle-specific gene associated with endometrial breakdown. Using the mRNA transcripts from MMP 11, NASBA could be utilized for menstrual blood identification. In this study, non-menstrual and menstrual blood samples were analyzed with NASBA both in the presence and absence of chemical contamination. Contaminants utilized ranged from commercial automotive wax, transmission fluid, brake fluid, artificial tears, hand soap, 10% bleach, and the luminol blood detecting reagent. Non-menstrual blood was aliquoted onto a 1 cm x 1 cm cotton cloth for contamination, while menstrual blood was provided on a 1 cm x 1 cm area of sterile menstrual pad. All samples underwent Tri reagent extraction to obtain RNA samples for NASBA amplification.With respect to NASBA amplification data, non-menstrual blood data (from extracted RNA and unextracted blood samples) revealed the highest levels of amplification as shown in relative fluorescence units (RFU). Uncontaminated menstrual blood revealed the second highest amplification data. In the presence of chemical contamination, high levels of amplification were observed when samples were contaminated with brake fluid and commercial hand soap. Moderately low amplification was observed with samples contaminated with transmission fluid, 10% bleach, and artificial tears. NASBA amplification was completely inhibited in the presence of automotive wax and luminol. Cycle threshold (CO values for each amplification result were also obtained from each reaction. Smaller Ct values correspond to a higher NASBAreaction efficiency and therefore larger amplification values. The Ct values obtained for each amplified sample correlate strongly with the amount of amplification observed from reaction. Based upon the results of this experiment, NASBA should be considered as a novel tool for forensic evidence analysis. / Department of Biology