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Title: NMR characterisation of the conserved RNA motifs governing the 4-way junction of the EMCV and FMDV IRES elements
Author: Chan, Kwong
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2013
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Translation initiation of encephalomyocarditis virus (EMCV) and foot and mouth disease virus (FMDV) occurs by a novel cap-independent mechanism which involves a RNA sequence termed internal ribosomal entry site (IRES). The role of IRES involves the recruitment of 40S ribosomal subunit and associated protein factors which are essential for translation to initiate. However, the tertiary structure and interactions of IRES is not known so far and therefore, the mechanism is not completely understood. A highly conserved and thermodynamically stable apical hammerhead motif in the I domain of EMCV and domain 3 of FMDV IRES elements has been selected for NMR analysis and structural characterisation using a range of homo- and heteronuclear 2D NMR experiments. A 15mer motif of the EMCV hammerhead region was selected for detailed NMR analysis and structural characterisation and it resulted in a high resolution NMR solution structure of the 15mer RNA with low energy and RMSD. The structure consisted of five canonical base pairs and a pentaloop as opposed to the predicated six canonical base pairs and triloop. Mutational studies have shown that certain smaller motifs of the hammerhead region are essential for translation activity. One such motif is the 11mer with identical sequence in both EMCV and FMDV. RNA analysis of this small RNA motif revealed that it adopted two different conformations. However, this appeared to vanish as the 11mer was extended to a longer 28mer, which also comprised of the 15mer RNA (EMCV), and 25mer (FMDV) where only one conformation of the 11mer can be observed. The EMCV four-way junction which consisted of the EMCV 28mer was investigated. Clear formation of the four way junction was achieved through the NMR analysis of a 44mer hetero-duplex and a single stranded 48mer RNA where observation of imino proton-amino proton cross peaks indicated the formation of all four stems of the junction. Based on the above, tandem repeats of the four-way junction were designed which resulted in 44mer and 88mer homo-duplexes. The NMR analysis of all EMCV RNA motifs showed clear conservation of EMCV 15mer in terms of chemical shifts and NOE cross peak patterns. Selectively 19F-labelled (5-fluorouracil) EMCV RNA motifs of the EMCV 15mer, 28mer and the 44mer homo-duplex were also studied and it was highly successful in confirming the NMR assignment of the unlabelled RNA samples. In the case of the larger 44mer homo-duplex, the fluorine label also allowed the confirmation of the assignment of an overlapped resonance which led to the detection of connectivity between two nucleotides that was not observed before in the unlabelled sample. The NMR assignment of the conserved RNA motifs of EMCV and FMDV IRES has generated valuable information in understanding the properties of the motifs which led to the first characterisation of the EMCV four-way junction. To the best of my knowledge, this is the first direct NMR evidence that a stable four-way junction can be formed at the EMCV hammerhead region. The detailed NMR analysis of the various RNA motifs contributed to an increased understanding of the IRES cap-independent mechanism and should further the use of IRES in biotechnology including RNA nanomedicine.
Supervisor: Ramesh, Vasudevan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available