Eukaryotic genomes including the human contain tandem arrays of triplet repeats and subsets of these repeats are associated with the triplet expansion disorders. These repeats are the molecular basis of several human neurological or neuromuscular genetic diseases that display unusual genetic properties like Fragile X syndrome (1,2).
Why one should study Fragile X syndrome and why a “knock in” mouse is needed? Fragile X syndrome is a mental retardation disease associated with the expansion of unstable CGG repeats (>200) in the 5’ UTR of the FMR1 gene and happens to occur in men and women frequently. An in vitro cloning technique was used to construct a “Knock In” transgenic mouse, having 176 repeats of CGG (permutation size repeats) within the mouse FMR1 5’ UTR region. The generation of the mouse and propagation is still in process. The mouse will enable us to understand the mechanism of the expansion of the repeats from pre mutation to full mutation range.
How mammalian cells behave on exposure of the repeats? HEK293 cell lines were derive with either 176 CGG or 22 CGG repeats corresponding to permutation size and normal size respectively. The pre-mutation sized CGG-repeat cell line shows a significant increase in apoptosis independent of Caspase 3, a precursor protein for the apoptosis process, relative to cells with repeats in the normal range. Since the repeats were not transcribed in this cell-line, the effect we have seen is a direct consequence of the CGG repeats. The persistent elevated level of apoptosis may contribute to a decrease in the number of ova in female carriers of “pre-mutation” alleles and to the cerebellar degeneration seen in older male carriers (3)
CGG DNA repeats forms a complex mixture of hairpin and tetraplex structures. What happened to the CGG RNA repeats? Under physiological conditions (CGG)22 RNA rapidly forms a single stable hairpin with no evidence of other folded structures even at low pH. RNA with a sequence (CGG)9AGG(CGG)12AGG(CGG)97, found in the Fragile X permutation allele, forms three consecutive hairpins with a total DG = -267.3. CGG RNA does not activate PKR, an interferon induced protein kinase, however, this RNA is digested by Human dicer enzyme although inefficiently, a central step in the RNA interference effect on gene expression. This may account for the stalling of the 40S ribosomal subunit that is thought to contribute to the translation defect in fragile X pre-mutation and full mutation alleles (4).
If it is not a triplet repeats does it behave in the same way? Progressive myoclonus epilepsy type I is a cerebral ataxia resulting in loss of cerebellar granule cells. This disease does not involve triplet repeats instead it involves expansion of a tandem array of dodecamers. Synthetic oligonucleotides containing these repeat shows an i-motif formation in case of the C-rich top strand and an antiparallel G-tetraplex formation in the G-rich bottom strand which is highly stable in presence of K+ ion in physiological conditions. We hypothesized that a multiplex structure formation takes place in the promoter region, leading to instability of the repeats (5).
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presentation of these studies.
References:
1. Wells, R. D., and S. T. Warren (ed.). 1998. Genetic instabilities and hereditary neurological disorders. Academic Press, San Diego, Calif.
2. Ashley, C., Jr., and S. T. Warren. 1995. Trinucleotide repeat expansion and human disease. Annu. Rev. Genet. 29:703-728.
3. Saha T, Handa, V, Goldwater, D., and Usdin, K.; RNA’s from fragile X triplet repeats and polyglutamine tract repeats form hairpin structure under physiological condition. (In preparation).
4. Handa, V, Saha, T. and Usdin, K, The Fragile X syndrome repeats form hairpins that do not activate interferon-inducible protein kinase, PKR, but are cut by Dicer Nucl. Acid. Res. 2003, 31(21), 6243-6248.
5. Saha, T. and Usdin, K.; Tetraplex formation by the progressive myoclonus epilepsy type-1 repeat: Implications for instability in the Repeat expansion diseases. FEBS Letts. 2001, 491, 184-187.