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Title: RNA target recognition by IMP1 : a molecular investigation at the transcriptome level
Author: Gallagher, Christopher
ISNI:       0000 0004 7429 1920
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2018
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RNA binding proteins orchestrate the assembly of macromolecular RNA-protein complexes that regulate post-transcriptional regulation. Disruption of these finely tuned processes can result in an array of diseases. RNA binding proteins are multidomain proteins organised into a modular structure. It is through this structure that they can recognise a vast repertoire of RNA targets by employing combinatorial binding. However, this mechanism is poorly understood. The IMP1 protein provides a model system in which we can investigate how domain sequence specificity and combinatorial binding define in vivo RNA selection. IMP1 recognises RNA via six putative RNA binding domains (two N-terminal RNA recognition motifs and four C-terminal K-homology domains). To date, IMP1 is known to bind a diverse range of RNA targets, both in homeostatic cellular events and in cancer. However, detailed information as to how IMP1 recognises these targets, especially at the individual domain level in context of the full-length protein is lacking. I have implemented a structural driven mutational approach to modify the RNA recognition properties of the individual RNA binding domains of the IMP1 protein. I have successfully generated iCLIP libraries for mutant IMP1 proteins where each KH domain was mutated in turn to inhibit RNA binding. From comparative analysis of these data sets I have begun to explore in vivo RNA target selection at the individual domain level and observed an altered RNA binding pattern to the ACTB mRNA. I have investigated in vitro, the RNA sequence specificity of the KH3-KH4 and the RRM1-RRM2 domains. This has led to the design of a mutant which shifts the RNA specificity of the KH3 domain. In addition to the identification that the RRM domains of IMP1 and IMP3 specifically recognise different RNA target sequences with different affinities, and the key residues involved in RNA recognition. These findings will aid in the further understanding of IMP in vivo RNA target selection.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available