|M.Sc Student||Ofer Noa|
|Subject||Enhancement of the Translation of FMR1 Premutation mRNA by|
Tetraplex RNA Destabilizing Agents
|Department||Department of Medicine||Supervisor||Professor Michael Fry|
|Full Thesis text|
Fragile X Syndrome (FXS), the most common hereditary cause of mental retardation, results from expansion of a d(CGG)2-54 repeat sequence at the 5'-UTR of the first exon of the FMR1 gene. FXS carriers have premutation alleles that include >55 to 200 (CGG) repeats that are prone to greater expansion in future generations. A full mutation of >200 repeats results in FMR1 silencing and FXS. Synthesis of FMR1 mRNA increases 5 to 10-fold in cells of premutation carriers while the level of its product protein FMRP remains normal or slightly below normal. As a result, about 30% of the premutation male carriers develop Fragile X associated Tremor/Ataxia Syndrome (FXTAS) and ~20% of the female carriers develop Premature Ovarian Failure (POF). The working hypothesis of this study is that premutation r(CGG)>55-200 repeat in FMR1 mRNA form quadruplex structures that hinder the translation of FMRP. Therefore, by destabilizing the translation blocking quadruplex RNA, tetraplex destabilizing agents may elevate the efficiency of translation of FMR1 premutation mRNA. We report that the quadruplex d/r(CGG)n destabilizing cationic porphyrin TMPyP4 unwound in vitro a monomolecular quadruplex structure of r(CGG)33. Correspondingly, we showed that increasing amounts of TMPyP4 progressively diminished the efficiency of the in vitro translation of a reporter firefly (FL) mRNA that contained a 5'-UTR premutation r(CGG)99 tract. To investigate the capacity of TMPyP4 alone and in combination with the quadruplex (CGG)n destabilizing proteins CBF-A or hnRNP A2 to increase the efficiency of translation in vivo of premutation mRNA, these agents were introduced into HEK293 cells that were transfected with plasmids with a (CGG)n (n = 0, 30, 99) tract upstream to firefly luciferase (FL) reporter gene. We show that TMPyP4 in excess over FL-mRNA resulted in selective elevation of the efficiency of translation of r(CGG)99-FL-mRNA. When exposed to TMPyP4 together with an excess of CBF-A, the two agents cooperated to additively elevate the efficiency of the in vivo translation. Whereas low relative ratios of TMPyP4, CBF-A or hnRNP A2 each by themselves did not affect the efficiency of translation of premutation mRNA, introduction of TMPyP4 together with sub-saturating amounts of either CBF-A or hnRNP A2 yielded a synergistic increase in the efficiency of the in vivo translation of (CGG)99-FL-mRNA. We propose that by increasing the efficacy of translation of premutation FMR1 mRNA, destabilizing quadruplex r(CGG)n agents such as TMPyP4, CBF-A or hnRNP A2 may alleviate FXTAS and/or POF.