Project summary: The long-term goal of this proposal is to understand, in detail, the mechanisms of mammalian mRNA 3' processing and its regulation. mRNA 3'-end formation, typically involving an endonucleolytic cleavage followed by polyadenylation, is an essential step of eukaryotic gene expression and it significantly impacts many aspects of RNA metabolism, including mRNA stability, subcellular localization and translation. In addition, the majority of eukaryotic genes produce multiple mRNA isoforms with distinct 3' ends through alternative polyadenylation (APA). Recent studies have revealed that APA is highly regulated in development and plays an important role in post- transcriptional gene regulation. Aberrant APA patterns have been associated with a wide range of diseases, from cancer to neurological disorders. Two key outstanding questions in the mRNA 3' processing field have been: 1) what is the molecular mechanism of mRNA 3' processing (including mechanisms for poly(A) site (PAS) recognition and catalysis of cleavage and polyadenylation)? 2) how is PAS selection or APA regulated? To address these fundamental questions, it is essential to understand the structure-function relationship of mRNA 3' processing factors. In published studies during the previous funding periods, we have reconstituted key modules of the mammalian mRNA 3' processing complex, characterized how AAUAAA and the U/GU-rich downstream element (two key elements that define the majority of mammalian PAS) are recognized, and revealed that mRNA 3' processing and splicing can be regulated through a similar mechanism. Building on these findings, here we propose to define the structure-function relationship of the fully reconstituted human mRNA 3' processing complex and systematically characterize the role of RNA-binding proteins (RBPs) in regulating PAS selection and APA. !