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Title: Architecture of eukaryotic mRNA 3' end processing machinery and insights into the mechanism of polyadenylation
Author: Kumar, Ananthanarayanan
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Almost all eukaryotic messenger RNAs (mRNAs) have a polyadenosine (polyA) tail at their 3' end that is added by a multi-protein complex known as cleavage and polyadenylation factor (CPF). CPF, along with accessory cleavage factors (CF) IA and IB, cleaves the pre-mRNA within the 3' untranslated region (UTR) and adds a poly(A) tail. Previous work has shown that CPF is a fourteen-subunit complex that is organised into three enzymatic modules: phosphatase, nuclease and polymerase. The polymerase module consists of the poly(A) polymerase Pap1, three RNA binding proteins (Cft1, Pfs2 and Yth1) and an unstructured protein Fip1. To understand how polymerase module recognizes specific RNA elements and how polyA tail addition is coordinated with other factors, I recombinantly expressed and purified a five-subunit polymerase module. Electron cryomicroscopy analysis resulted in a 3.5 Å resolution structure of Cft1-Pfs2-Yth1, revealing four β propellers in an arrangement reminiscent of other nucleic acid binding complexes. Using biochemical assays, I show that CF IA stimulates polyadenylation activity of CPF by interacting with polymerase module and tethering it to substrate RNA. Thus polymerase module acts as a hub to bring together the RNA, Pap1 and cleavage factors for specific and efficient polyadenylation. The poly(A) tail length of newly made pre-mRNAs in S. cerevisiae is ~ 60 As. The nuclear poly(A) binding protein Nab2 is known to have a role in poly(A) tail length control. The molecular mechanism behind how CPF terminates polyadenylation to regulate uniform poly(A) tail length remains elusive. Using an in vitro polyadenylation assay with highly pure protein complexes, I have studied the mechanism of poly(A) tail length control by CPF. The assays highlight the contribution of the cleavage factors and the phosphatase module of CPF towards regulating the poly(A) tail length of a substrate RNA. Taken together, the findings discussed in this dissertation provide new insights into the architecture of eukaryotic mRNA 3' end processing machinery and into the mechanism of polyadenylation by CPF.
Supervisor: Passmore, Lori Sponsor: GATES Cambridge
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: mRNA ; Poly(A) tail ; Cryo-EM ; CPF ; Gene regulation