This dissertation focuses on pre-mRNA 3'-end processing in eukaryotes, a crucial step in defining the 3'end of most protein-coding mRNAs. In vertebrates, two distinct molecular machines are involved: the canonical machinery, consisting of a Cleavage Factor (CF) module, Polyadenylation Specificity (PSF) module, Cleavage Stimulation Factor, and other complexes, and the U7 snRNP machinery (U7 machinery), which consist of a core U7 snRNP complex and the Histone Cleavage Complex (HCC). U7 snRNP is involved in replication-dependent histone pre-mRNA 3'-end processing. Interestingly, the cleavage modules of the canonical and U7 machinery share an endonuclease, CPSF73, that catalyzes the cleavage reaction for 3’-end processing of pre-mRNAs. CPSF73 also possesses 5’-3’ exonuclease activity in the U7 machinery. CPSF73 has been identified as a potential target for anticancer and antimalarial small-molecule inhibitors. Traditionally, CPSF73 nuclease activity has been demonstrated using a gel-based end-point assay, using radio-labeled or fluorescently labeled RNA substrates. In Chapter Two (Ch. 2) of this dissertation introduces a novel, real-time fluorescence assay to investigate CPSF73 nuclease activity. This efficient and high-throughput assay holds potential for identifying new CPSF73 inhibitors. Chapter Three (Ch. 3) of this dissertation delves into the structural characterization of the mammalian PSF (mPSF) module in complex with the second most frequent PAS variants, AUUAAA. Structure studies have revealed the molecular mechanism underlying mPSF recognition of the most common PAS sequence, AAUAAA. This study presents a cryo-EM structure of mPSF in complex with AUUAAA. While the binding modes remain highly similar between the two PAS variants, we observed conformational differences in the A1 and U2 nucleotides in AUUAAA compared to the A1 and A2 of AAUAAA. Furthermore, CPSF30 displayed conformational changes near the U2 nucleotide of AUUAAA. Attempts to explore the binding modes of two rare PAS sequences, AAGAAA and GAUAAA, were inconclusive due to a lack of RNA density in the EM maps. An atomic model of the ternary structure (CPSF160, WDR33, CPSF30) was produced using the EM map of the AAGAAA sample. The ternary structure revealed PAS recognizing residues to be disordered in CPSF30 (ZF2 and ZF3) and WDR33. Overall, this dissertation provides insights into the intricate mechanisms of pre-mRNA 3'-end processing in mammals, laying the groundwork for future studies and potentially leading to the development of novel inhibitors targeting CPSF73.