Investigation of post-transcriptional regulation of c-myc mRNA by RNA binding proteins (Doctoral thesis)

Λαμπριανίδου, Ανδρομάχη

Aberrant expression of c-MYC is involved in many stages of carcinogenesis. Thus, its expression requires tight control on many levels including post transcriptional regulation. c-myc mRNA carries within the coding region a 249 nucleotide sequence (Coding Region instability Determinant -CRD) that confers instability and suppresses translation. This sequence is recognized by the mRNA binding protein IMP1, which stabilizes c-myc mRNA, protecting it from endonucleases. IMP1 is expressed in fetal tissues and de novo in malignancies. It is phosphorylated at Tyr 396 by Src kinase and at Ser 181 by mTORC2, respectively. In this study, the possible effect of these modifications upon the expression of c-MYC was investigated. The translatability of the chimaeric transcripts carrying the CRD c-myc sequence, in frame, was reduced by 35% in the presence of the Src kinase inhibitor (SrcI1) although the corresponding levels of mRNA were increased. Chimaeric luciferase plasmids carrying the CRD c-myc sequence, were co-transfected with plasmids expressing the wild-type IMP1 protein (IMP1wt) or its mutant counterpart (IMP1Y396F). IMP1wt reduced translatability by 20% while over-expression of the mutant protein by 35%. Inhibition of Src activity in the presence of IMP1wt further reduced the translatability of the chimaeric transcripts. IMP1Y396F or IMP1wt in the presence of the Src inhibitor, were detected in cytoplasmic granules. Under these conditions, the endogenous c-myc mRNA was stabilized, although it was not translated indicating that in the absence of Src signaling IMP1 stored reversibly c-myc mRNA in cytoplasmic granules.Inhibition of mTORC2 signaling resulted in the destabilization of chimaeric transcripts as well as of the endogenous c-myc mRNA, 20% and 32%, respectively. However, an increase of 80% in c-ΜΥC protein levels was observed in the presence of the Torin1 inhibitor, while the presence of the IMP1S181A protein doubled c-ΜΥC levels and caused apoptosis in 23% of the cells.In order to augment c-ΜΥC expression and enhance apoptosis, sequential inhibition of IMP1 phosphorylation by the two pathways was applied. The accumulated ''stored'' c-myc mRNA, due to the presence of SrcI1 for 48 hours, after the subsequent exposure of the cells to the inhibitor Torin1 yielded increased protein levels of both the chimaeric transcripts carrying the CRD sequence and the endogenous c-ΜΥC, 2.5 and 3 fold, respectively. Such an increase in c- MYC levels caused apoptosis in 44% of the cell population.Finally, the effect of the sequential action of the Src kinase inhibitor AZD0530 and Torin1, upon HeLa cell xenografts was evaluated. Administration of the AZD0530 inhibitor did not cause any change in tumor growth rate. However, the prior exposure of tumor cells to AZD0530 enhanced the effect of Torin1. The sequential use of the two substances resulted in statistically significant inhibition of the xenograft growth compared to those exposed to Torin1 alone.
Institution and School/Department of submitter: Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Βιολογικών Εφαρμογών και Τεχνολογιών
Subject classification: Πρωτεΐνες -- Χημεία
Keywords: Πρωτεΐνη c-ΜΥC,Πρωτεΐνη IMP1,Μετά - μεταγραφική ρύθμιση,Μεταφρασιμότητα,Kινάση c - Src,Κινάση mTORC2,Μετα - μεταφραστική τροποποίηση,Φωσφορυλίωση,Απόπτωση,Κυτταροπλασματικά κοκκία,Βλάβες στο DNΑ,Ξενομοσχεύματα,c-myc mRNA,c-ΜΥC protein,IMP1 protein,Post trancriptional regulation,Stability and translatability of mRNA,c-Src kinase,mTORC2 kinase,Post translational modifications,Phosphorylation,Apoptosis,Cytoplasmic granules,DNA damage,Xenografts
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