Chemical and Structural Probing of the N-Terminal Residues Encoded by FMR1 Exon 15 and Their Effect on Downstream Arginine Methylation

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• 作者：Natalia Dolzhanskaya ; David C. Bolton ; Robert B. Denman
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：August 19, 2008
• 年：2008
• 卷：47
• 期：33
• 页码：8491-8503
• 全文大小：570K
• 年卷期：v.47,no.33(August 19, 2008)
• ISSN：1520-4995

文摘

Exon 15 of the fragile X mental retardation protein gene (FMR1) is alternatively spliced into three variants. The amino acids encoded by the 5′ end of the exon contain several regulatory determinants including phosphorylation sites and a potential conformational switch. Residues encoded by the 3′ end of the exon specify FMRP’s RGG box, an RNA binding domain that interacts with G-quartet motifs. Previous studies demonstrated that the exon 15-encoded N-terminal residues influence the extent of arginine methylation, independent of S₅₀₀ phosphorylation. In the present study we focus on the role the putative conformational switch plays in arginine methylation. Chemical and structural probing of Ex15 alternatively spliced variant proteins and several mutants leads to the following conclusions: Ex15c resides largely in a conformation that is refractory toward methylation; however, it can be methylated by supplementing extracts with recombinant PRMT1 or PRMT3. Protein modeling studies reveal that the RG-rich region is part of a three to four strand antiparallel β-sheet, which in other RNA binding proteins functions as a platform for nucleic acid interactions. In the Ex15c variant the first strand of this sheet is truncated, and this significantly perturbs the side-chain conformations of the arginine residues in the RG-rich region. Mutating R₅₀₇ in the conformational switch to K also truncates the first strand of the β-sheet, and corresponding decreases in in vitro methylation were found for this and R₅₀₇/R₅₄₄ and R₅₀₇/R₅₄₆ double mutants. These effects are not due to the loss of R₅₀₇ methylation as a conformational switch-containing peptide reacted under substrate excess and in methyl donor excess was not significantly methylated. Consistent with this, similar changes in β-sheet structure and decreases in in vitro methylation were observed with a W₅₁₃-K mutant. These data support a novel model for FMRP arginine methylation and a role for conformational switch residues in arginine modification.