|M.Sc Student||Hershco-Shani Inbal|
|Subject||Destabilization of Tetrahelical Transcripts of the Fragile X|
Syndrome (CGG)n Expanded Sequence by Members of
the hnRNP Family and by Cationic
|Department||Department of Medicine||Supervisors||? 18? Avram Hershko|
|Professor Michael Fry|
Fragile X syndrome, the most common inherited cause of mental retardation, is commonly caused by a substantial expansion of a d(CGG)n trinucleotide repeat sequence in the 5’ untranslated region of the first exon of the X-linked housekeeping gene Fragile X Mental Retardation 1 (FMR-1) that encodes FMRP protein. Experimental results indicated that d/r(CGG)n tracts readily fold into hairpin structures and assemble to form tetrahelical structures. These secondary structures are thought to cause expansion of the repeat sequence and to block the translation of FMR-1 mRNA. The ability of different agents to diminish the generation and the stability of hairpin or tetraplex structures of the (CGG) trinucleotide repeat in DNA or RNA may contribute to the restoration of the FMR1 transcription and to synthesis of FMRP in cells of individuals affected with fragile X syndrome.
In the first part of this work we show that r(CGG)n forms in a K+ dependent reaction a mixture of four-stranded mono- and multimolecular structures that are stabilized in part by Hoogsteen hydrogen bonds.
The cationic porphyrin TMPyP4 was shown in the past to unfold tetraplex structure of d(CGG)7 whereas TMPyP2 and TMPyP3 were devoid of such destabilizing activity. In the second part of this work we report that the ability of TMPyP4 to stabilize or destabilize multimolecular tetraplex structures of d / r(CGG)n depends on the number of (CGG) repeats units and that TMPyP2 and TMPyP3 increased or did not affect the thermal stability of r(CGG)n.
hnRNP-related proteins, CBF-A and hnRNP A2, were found to be capable of destabilization of bimolecular d(CGG)7 by conserved motifs, RNP11 element and the ATP/GTP binding box, whereas the RNP21 element blocked their unwinding activity. In the third part of this work we report that G4 r(CGG)7 was effectively destabilized by hnRNP A2 but not by CBF-A. The tetraplex RNA disrupting activity of hnRNP A2 was mediated by the same conserved domains that execute destabilization of bimolecular d(CGG)n. We also show that the ability of hnRNP A2 to destabilize multimolecular structures of RNA or DNA that contain 33 (CGG) repeats depended on their chemistry, whether they are multimolecular structures of DNA or RNA.