ABSTRACT
Hairpin or tetrahelical structures formed by a d(CGG)n sequence in the FMR1 gene are thought to promote expansion of the repeat tract. Subsequent to this expansion FMR1 is silenced and fragile X syndrome ensues. The injurious effects of d(CGG)n secondary structures may potentially be countered by agents that act to decrease their stability. We showed previously that the hnRNP-related protein CBF-A destabilized G'2 bimolecular tetraplex structures of d(CGG)n. Analysis of mutant proteins revealed that the CBF-A-conserved domains RNP11 and ATP/GTP binding box were sufficient and necessary for G'2 d(CGG)n disruption while the RNP21 motif inhibited the destabilization activity. Here, we report that a C-terminal fragment of CBF-A whose only remaining conserved domain was the ATP/GTP binding motif, disrupted G'2 d(CGG)n more selectively than wild-type CBF-A. Further, two additional members of the hnRNP family, hnRNP A2 and mutant hnRNP A1 effectively destabilized G'2 d(CGG)n. Examination of mutant hnRNP A2 proteins revealed that, similar to CBF-A, their RNP11 element and ATP/GTP binding motif mediated G'2 d(CGG)n disruption, while the RNP21 element blocked their action. Similarly, the RNP11 and RNP21 domains of hnRNP A1 were, respectively, positive and negative mediators of G'2 d(CGG)n destabilization. Last, employing the same conserved motifs that mediated disruption of the DNA tetraplex G'2 d(CGG)n, hnRNP A2 destabilized r(CGG)n RNA tetraplex.
Subject(s)
DNA/chemistry , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/chemistry , Nerve Tissue Proteins/genetics , RNA-Binding Proteins , Adenosine Triphosphate/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Conserved Sequence , Fragile X Mental Retardation Protein , G-Quadruplexes , Guanosine Triphosphate/metabolism , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , Protein Structure, Tertiary , RNA/chemistry , RNA Stability , Repetitive Sequences, Nucleic Acid , Sequence Homology, Amino AcidABSTRACT
Fragile X syndrome, the most common cause of inherited mental retardation, is instigated by dynamic expansion of a d(CGG) trinucleotide repeat in the 5'-untranslated region of the first exon of the FMR1 gene, resulting in its silencing. The expanded d(CGG)(n) tract readily folds into hairpin and tetraplex structures which may contribute to the blocking of FMR1 transcription. In this work, we report that the cationic porphyrin 5,10,15,20-tetra(N-methyl-4-pyridyl)porphin (TMPyP4) effectively destabilizes in vitro the G'2 bimolecular tetraplex structure of d(CGG)(n) while it stabilizes the G'2 tetraplex form of the telomeric sequence d(TTAGGG)(2). Similarly to TMPyP4, the hnRNP-related protein CBF-A also destabilizes G'2 tetrahelical d(CGG)(n) while binding and stabilizing tetraplex telomeric DNA. We report that relative to each agent individually, successive incubation of G'2 d(CGG)(n) with TMPyP4 followed by exposure to CBF-A results in a nearly additive extent of disruption of this tetraplex form of the repeat sequence. Our observations open up the prospect of unfolding secondary structures of the expanded FMR1 d(CGG)(n) tract of fragile X cells by their exposure to low molecular size drugs or to proteins such as TMPyP4 or CBF-A.
