Investigation of the PABPC1-BTG2 interaction and its effects on mRNA deadenylation by CNOT7(CAF1)

Ameerul Moominin, Aalam Mohammed (2022) Investigation of the PABPC1-BTG2 interaction and its effects on mRNA deadenylation by CNOT7(CAF1). PhD thesis, University of Nottingham.

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Abstract

Polyadenylation is the process of the addition of a poly(A) tail to the 3ʹ end of mRNA. The binding of human poly(A) binding protein to the poly(A) tail can either initiate or repress translation or regulate mRNA degradation. Cytoplasmic mRNA degradation in eukaryotes involves shortening of mRNA poly(A) tail by poly(A)-selective ribonuclease (deadenylase) enzymes. A recent study reported that PABPC1 could stimulate deadenylation by CNOT7/CAF1, a catalytic subunit of the CCR4-NOT complex, in the presence of BTG2. BTG2 is antiproliferative, regulating cell cycle progression in various cell types. BTG2 bridges PABPC1 and CNOT7/CAF1 and regulates deadenylation. This study aims to understand how PABPC1 and BTG2 interact, starting by identifying the amino acid residues of PABPC1 and BTG2 that contribute to the interaction.

The surface conserved PABPC1 residues predicted to interface with BTG2 were identified. Since PABPC1 has RNA bound to its structure, the expected PABPC1 interaction surface residues were compared with known PABPC1 residues interfacing with poly(A) RNA. Comparing residues led to identifying PABPC1 candidate residues free from RNA interfaces and residues required for interaction with BTG2. Subsequently, from the identified candidate residues, data-driven docking was used to generate a computational model depicting PABPC1-BTG2 interaction. The generated PABPC1-BTG2 model was used to create a quaternary model for poly(A) RNA-PABPC1-BTG2-CNOT7/CAF1. The quaternary model revealed that the 3ʹ end of the poly(A) RNA is accessible for the active site of CNOT7/CAF1. Site-directed mutagenesis studies were consequently conducted by introducing point mutations to candidate residues of PABPC1 and residues in Box C of BTG2. Subsequently, pull-down assays were performed to determine several amino acids residues of PABPC1 required for interaction with BTG2.

Further investigation of RNA binding for PABPC1/variants revealed that the PABPC1 residues necessary for interaction with BTG2 do not interfere with poly(A) RNA binding. The quaternary poly(A)-PABPC1-BTG2-CAF1 model created was validated by performing a functional assay. The functional assay revealed poly(A) RNA 3' end situated towards CNOT7/CAF1 catalytic centre suggests a rationale for enhanced deadenylation by CNOT7/CAF1 in the presence of BTG2 and PABPC.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Winkler, Sebastiaan
Falcone, Franco
Keywords: PABPC1-BTG2, mRNA deadenylation, CNOT7(CAF1)
Subjects: R Medicine > RM Therapeutics. Pharmacology
Faculties/Schools: UK Campuses > Faculty of Science > School of Pharmacy
Item ID: 68944
Depositing User: Ameerul Moominin, Aalam
Date Deposited: 28 Jul 2022 04:40
Last Modified: 28 Jul 2022 04:40
URI: http://eprints.nottingham.ac.uk/id/eprint/68944

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