Kiri, Arpna; (2000) The isolation and function of the 3'untranslated region of the myosin heavy chain genes of skeletal muscle. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In recent years the 3'untranslated regions (3'UTRs) of eukaryotic mRNAs have emerged as a repository of signals that determine mRNA localization, stability, translation, and cytoplasmic polyadenylation. Furthermore, these regions bind transacting factors to form specific complexes that control gene expression at the post-transcriptional level. This thesis describes the investigations of the functional role of the 3'untranslated region of four murine skeletal myosin heavy chain (MyHC) mRNAs. 3' RACE was used firstly to isolate and clone the slow type 1, and fast 2a, 2b, and 2x MyHC 3'UTRs. Sequence analysis revealed not only the isotypic conservation of the 3'UTRs but also two conserved motifs in the fast 3'UTRs which could be involved in regulation of gene expression or act as putative binding sites for proteins. The results of transfection experiments with hybrid CAT/MyHC 3'UTRs plasmids showed that, after taking into account the effect of deleting the CAT 3'UTR, the MyHC 3'UTRs had no major effects on translation and stability of reporter mRNA in transfected C2C12 cells. RNA-protein interactions of the MyHC 3'UTRs were investigated by bandshift analyses. Results demonstrated that the MyHC 3'UTR-protein complexes were muscle-specific. Competition assays to determine sequence specificity showed that binding could be competed out by unlabelled MyHC 3'UTR and polyadenylic acid but not rat growth hormone 3'UTR. Deletion analysis suggests that secondary structure is important for protein binding. Purification of the protein from shifted complexes identified a 33-kDa protein, aldolase A, as a MyHC 3'UTR-binding protein. These results and the role of aldolase A in the post-transcriptional regulation of MyHC are discussed.

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