Binding interactions of the mRNA regulator CELF1

Edwards, John Michael (2013) Binding interactions of the mRNA regulator CELF1. PhD thesis, University of Nottingham.

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CELF1 is an RNA binding protein with regulatory roles in translation, alternative splicing and mRNA degradation. This protein is of particular interest as its upregulation is believed to be involved in the pathogenesis of type 1 myotonic dystrophy. CELF1 functions by binding to a specific sequence in the 3’ untranslated region of its target mRNAs. This sequence has been termed the “EDEN” motif, but the exact requirements for binding of CELF1 were not well defined. In this study we therefore aimed to determine the sequence requirements for an RNA substrate to form a high affinity interaction with CELF1, and characterise the structure of the resulting complex. The CELF1 protein is composed of three structured RNA recognition motifs separated by flexible linkers. Our strategy was to investigate the RNA binding properties of each domain in isolation, and then the requirements for tandem binding of the domains in order to build up the complete “EDEN” motif capable of forming a high affinity complex with the wild type protein.

This has been accomplished using NMR spectroscopy to map the chemical shift perturbations in each domain on binding to a range of RNA substrates. ITC was also used to investigate the binding affinities of each domain, and the enhancement of affinity when domains bind in tandem. By these methods we have refined the sequence requirements for simultaneous binding of all domains of CELF1, and designed RNA substrates which will bind with higher affinity than any previously reported. We have also shown the potential involvement of RNA secondary structure in forming the CELF1 binding site, and identified two possible examples of this in natural mRNA targets.

CELF1 binding triggers deadenylation of its target mRNAs and this is suspected to be via a mechanism involving recruitment of poly (A) ribonuclease. These two proteins have been shown to interact, but no structural information was available to show which domains were interacting, or whether CELF1 was capable of forming a ternary complex with both RNA and poly(A) ribonuclease. Since the ribonuclease exists as a 146 kDa dimer, the complex of it with CELF1 was an ambitious target for NMR. In this study we demonstrate that high resolution NMR data can be acquired on this key regulatory complex. Using this we go on to confirm the interaction between these two proteins, and that the domains involved in binding suggest a ternary complex is possible.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Searle, M.S.
Soultanas, P.
Subjects: Q Science > QP Physiology > QP501 Animal biochemistry
Faculties/Schools: UK Campuses > Faculty of Science > School of Chemistry
Item ID: 13453
Depositing User: EP, Services
Date Deposited: 10 Mar 2014 15:13
Last Modified: 17 Dec 2017 06:00

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