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Structural and functional characterisation of the cold-inducible RNA-binding protein CIRP and its application to enhanced recombinant protein production

Creigh-Pulatmen, Tilbe (2014) Structural and functional characterisation of the cold-inducible RNA-binding protein CIRP and its application to enhanced recombinant protein production. Doctor of Philosophy (PhD) thesis, University of Kent,. (KAR id:47913)

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Expression of the RNA binding protein CIRP is up regulated in mammalian cells upon perception of mild cold shock (27-32°C), conditions that can result in enhanced recombinant protein yields from mammalian cells and improved protein folding and activity. CIRP also binds to key proteins involved in the control of mRNA translation initiation, potentially acting as a bridge between the RNA and protein synthesis machinery. CIRP has two domains, an N terminal RNA binding domain and an arginine/glycine rich C-terminal domain that is natively disordered. The N-terminal domain includes two RNA-binding sites, RNP1 and RNP2 that are conserved across many RNA binding proteins. Here, the RNA binding of the N-terminal domain of CIRP was investigated by introducing mutations into the RNP1 and RNP2 RNA binding sites and monitoring subsequent RNA binding using electromobility shift assays and NMR. These studies show that the F49 and F9 residues in these regions are important for RNA interactions. Further, NMR dynamics studies showed that the region (?2-?3 loop) just before the RNP1 sequence that includes the F49 residue has increased motion compared to the rest of the protein. Chemical shift analysis was used to map those residues in CIRP involved in RNA binding that mapped onto the RNP1 and RNP2 sites. Mutation of the F49 and F9 sites to Ala residues disrupted RNA binding as shown by NMR studies. Mutation of each residue resulted in some conformational change in the structure of the domain as determined by HSQC-NMR, particularly for the F49 residue in RNP1. The mutation of one of the phenylalanine residues affected the chemical shift of the other, confirming their proximity in space. The C-terminal is a natively disordered domain but the studies presented here suggest this plays a role in RNA binding and ligand specificity. Cell lines stably expressing CIRP were also generated to further investigate the function of CIRP and to determine binding partners. CIRP was found to interact with the translation initiation factor eIF4G, both the full length CIRP and N-CIRP molecule, suggesting that binding to 4G occurs through the N-terminal domain. The over expression of CIRP enhanced recombinant firefly luciferase expression when the luciferase mRNA contained a CIRP 3’UTR binding sequence. CIRP over-expression also enhanced growth of Chinese hamster ovary cells at 37°C but not at reduced temperature. From these studies it is hypothesised that CIRP can bind specific mRNAs and enhance their expression through its interaction with both the specific mRNA and the translation machinery via eIF4G leading to increased protein expression of the target mRNA under conditions of mild cold-stress (32°C).

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Smales, Christopher Mark
Thesis advisor: Williamson, Richard
Thesis advisor: Howard, Mark
Uncontrolled keywords: Biochemistry, Protein Structure, Bioprocessing, Translation, Recombinant Protein Expression, Cold Stress, RNA Binding Proteins, Protein Interactions, Cold Shock Proteins, NMR, Protein Purification, Mutagenesis, Protein Solubility, Intrinsically Disordered Proteins, Protein Dynamics
Subjects: Q Science > QP Physiology (Living systems) > QP517 Biochemistry
Divisions: Divisions > Division of Natural Sciences > Biosciences
Funders: Organisations -1 not found.
Depositing User: Users 1 not found.
Date Deposited: 09 Apr 2015 11:00 UTC
Last Modified: 08 Dec 2022 13:14 UTC
Resource URI: (The current URI for this page, for reference purposes)

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Creigh-Pulatmen, Tilbe.

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