Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.556550
Title: Investigating the protein and RNA interactions of the Polypyrimidine Tract Binding protein : high resolution structures with implications in the regulation of alternative splicing
Author: Joshi, Amar
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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Abstract:
Alternative splicing produces multiple mRNAs from a single gene. It is a powerful mechanism used to amplify complexity of the metazoan proteome. The Polypyrimidine Tract Binding protein (PTB) is a key regulator of alternative splicing. In addition, it has roles in the localization and stability of mRNA and is involved in IRES mediated translation initiation. It has four RNA recognition motif domains (RRMs) which consist of two α‐helices which pack against a β‐sheet. The RRMs act in concert to direct splicing as each RRM binds pyrimidine rich RNA (UCUU or CUCUCU) across the β‐sheet. Solution structures have been solved of each RRM in complex with short hexameric oligonucleotides. However, high‐resolution structural studies of larger protein fragments with longer RNAs have been very difficult. Small angle X‐ray scattering has been used to probe this, but sample aggregation obstructed collection of high quality data. To address this we have investigated how longer RNAs bind PTB fragments. We have determined optimal RNAs which should help to determine structures of PTB fragments in complex with longer RNA molecules. PTB is necessary for skipping Tpm1 exon 3 in smooth muscle cells. However, interaction with an additional protein, Raver1, is required to accomplish this. Raver1 contains four PTB‐Raver1 interaction motifs (PRIs) with a consensus of [S/G][I/L]LGxxP. The PRIs bind to the α‐helical side of PTB RRM2. We have created chimeric fusion constructs where Raver1 PRIs are encoded as N‐terminal extensions to PTB RRM2. High‐resolution crystal structures have been solved of 2 PRIs bound to RRM2, one high affinity and one low affinity. The differences between the conformations of each PRI bound to the RRM explain differences in affinity. We have used structure‐based mutagenesis to probe these differences and have revealed what is necessary for a high affinity interaction. From this we have proposed a new consensus for high affinity PRIs [S/G][I/L]LGx[AVP]P Additionally, we have solved the structure of RRMs 3 and 4 of a neuronal homologue of PTB, nPTB. As with the prototypic protein, the helical sides of RRMs 3 and 4 interact forming a di‐domain with RNA binding surfaces on opposing sides.
Supervisor: Curry, Stephen ; Matthews, Steve Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.556550  DOI: Not available
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