Distinct domains in the CArG-box binding factor A destabilize tetraplex forms of the fragile X expanded sequence d(CGG)n.
Formation of hairpin or tetraplex structures of the FMR1 gene d(CGG)n sequence triggers its expansion, setting off fragile X syndrome. In searching for proteins that destabilize d(CGG)n secondary structures we purified from rat liver quadruplex telomeric DNA binding protein 42 (qTBP42) that disrupts G'2 bimolecular tetraplex d(CGG)n while paradoxically stabilizing the G'2 structure of the telomeric sequence d(TTAGGG)n. Based on peptide sequence homology of qTBP42 and mouse CArG-box binding factor A (CBF-A), we provide direct evidence that recombinant CBF-A protein is physically and immunochemically indistinguishable from qTBP42 and that it too destabilizes G'2 d(CGG)n while stabilizing G'2 d(TTAGGG)n. We inquired whether CBF-A employs the same or different domains to differentially interact with G'2 d(CGG)n and G'2 d(TTAGGG)n. Mutant CBF-A proteins that lack each or combinations of its five conserved motifs: RNP1(1), RNP1(2), RNP2(1), RNP2(2) and ATP/GTP-binding box were tested for their G'2 d(CGG)n destabilization and G'2 d(TTAGGG)n stabilization activities. We find that either RNP1(1) or the ATP/GTP motifs are necessary and sufficient for G'2 d(CGG)n destabilization whereas RNP2(1) suppresses destabilization by either one of these two motifs. Neither RNP1(1) nor the ATP/GTP motif are required for G'2 d(TTAGGG)n stabilization. Hence, CBF-A employs different domains to destabilize G'2 d(CGG)n or stabilize G'2 d(TTAGGG)n.
Unit of Biochemistry, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, PO Box 9649, Haifa 31096, Israel.
Pnina
P
Weisman-Shomer
Esther
E
Cohen
Michael
M
Fry
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051860:Fragile X Messenger Ribonucleoprotein 1; D054856:G-Quadruplexes; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D006358:Hot Temperature; D007106:Immune Sera / Q000276:immunology; D051379:Mice; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D016601:RNA-Binding Proteins; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012261:Ribonucleoproteins; D014157:Transcription Factors; D018911:Trinucleotide Repeats / Q000235:genetics*
D018797:Cell Cycle Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D051860:Fragile X Messenger Ribonucleoprotein 1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D007106:Immune Sera; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D012261:Ribonucleoproteins; D014157:Transcription Factors; C497684:Fmr1 protein, mouse; C497685:Fmr1 protein, rat; C121363:Hnrnpab protein, rat; C077048:Hnrnpab protein, mouse