טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentKhateb Samer
SubjectDestabilization of Tetraplex Structures of the Fragile X
Syndrome Expanded Sequence (CGG)n by
Members of the hnRNP Family
DepartmentDepartment of Medicine
Supervisors Professor Michael Fry
Dr. Pnina Shomer
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Fragile X syndrome (FX), the most common inherited cause of mental retardation, is initiated by dynamic expansion of a d(CGG) trinucleotide repeat in the 5’ untranslated region (5' UTR) of the first exon of the Fragile X Mental Retardation 1 (FMR1) gene at Xq27.3.


Stable FMR1 d(CGG)n runs in normal individuals have 7-54 d(CGG) repeats and fully affected individuals have >200-2000 d(CGG) repeats that cause FX. Premutaion carriers have >55-200 d(CGG) copies. One third of the premutated males present Fragile X associated Tremor/Ataxia Syndrome (FXTAS) and a fifth of the females have Premature Ovarian Failure (POF).


We hypothesized that premutation r(CGG)55-200 tract in FMR1 mRNA in carriers folds into tetrahelical structures that block FMRP translation. Second, we proposed that agents that destabilize the (CGG)n tetraplex structures in FMR1 mRNA may restore full synthesis of FMRP in FX carriers.

 

In the first part of this work we prepared proteins that disrupt tetraplex structures of (CGG)n in DNA and RNA. We show that members of the hnRNP family, CBF-A, hnRNP A2 and mutant hnRNP A1, destabilize G’2 d(CGG)n and identify in all three proteins conserved motifs that mediate G’2 d(CGG)n disruption. A mini protein consisting of the C-terminal 75 amino acids of CBF-A is shown to also destabilizes G'2 d(CGG)n.


In a second part of this work we explored the effects of expanded d/r(CGG)n repeats on transcription and translation in vitro. Employing transcription and translation reticulocyte lysate systems, we show that whereas 62 or 99 d(CGG)n premutation repeats minimally affect the transcription of a reporter Firefly Luciferase (FL) gene, 62 or 99 r(CGG) repeats fold into tetraplex structures that impede FL mRNA translation.


Last, we show that premutation-size (CGG)n tracts obstruct translation in HEK256 cells and that tetraplex disrupting hnRNPs alleviate this block. A (CGG)99 stretch depressed the efficiency of the in vivo translation of FL mRNA and active hnRNP A2 or CBF-A but not their respective inactive mutants alleviated the block to translation.


We conclude that some of the pathologies associated with FX permutation may be due to the over-production of ineffective FMR1 mRNA and that agents that resolve secondary structures of the expanded (CGG)n RNA tract may serve to allay clinical outcomes of the premutation.