The molecular basis of mRNA 3'-end processing

Poly-adenosine (poly(A)) tails are found at the 3'-end of almost every eukaryotic mRNA and play essential roles in regulating gene expression. Poly(A) tails are required for export of mRNAs from the nucleus, for efficient translation and for mRNA stability. They are added by the evolutionarily-conserved cleavage and polyadenylation factor (CPF/CPSF), a megadalton multi-subunit complex that cleaves pre-mRNAs, adds a poly(A) tail of specified length and promotes transcription termination. The activities of CPF (endonuclease, polymerase and phosphatase) must be intimately coupled with each other and with transcription but the molecular basis for this remains unknown. In this proposal, I will use an integrated approach to obtain a molecular understanding of eukaryotic mRNA 3'-end processing. Specifically, I aim to: 1) Determine the sequence specificity of the 3'-end processing machinery; 2) Understand the molecular basis of pre-mRNA recognition, cleavage and polyadenylation; 3) Understand how dynamics and conformational changes contribute to CPF activities; 4) Determine how CPF is coupled to transcription. In addition to cryoEM, X-ray crystallography, biochemical reconstitutions and functional studies, we will employ NMR and single-molecule studies to investigate the dynamics of this process. Together, this will define the molecular basis of mRNA 3'-end processing.