Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.643508
Title: Structural and functional characterisation of the cold-inducible RNA-binding protein CIRP and its application to enhanced recombinant protein production
Author: Creigh-Pulatmen, Tilbe
ISNI:       0000 0004 5354 4685
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2014
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Abstract:
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 (β2-β3 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).
Supervisor: Smales, Mark; Williamson, Richard; Howard, Mark Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.643508  DOI: Not available
Keywords: QP517 Biochemistry
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