Using capillary electrophoresis to study methylation effect on RNA-peptide interaction

• Autorzy: Mucha P. , Szyk A. , Rekowski P. , Agris P.F.
• Języki publikacji: EN

Abstrakty

EN

Methylation of RNA and proteins is one of a broad spectrum of post-trans- criptional/translational mechanisms of gene expression regulation. Its functional signification is only beginning to be understood. A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualitative study of the methylation effect on biomolecules interaction is presented. Two RNA-peptide systems were chosen for the study. The first one consists of a 17-nucleotide analogue (+27-+43) of the yeast tRNA Phe anticodon stem and loop domain (ASL Phe) containing three of the five natu­rally occurring modifications (2'-O-methylcytidine (Cm32), 2'-O-methylguanine (Gm34) and 5-methylcytidine (m5 C40)) (ASL Phe -Cm32,Gm34,m5 C40) and a 15-amino-acid peptide (named tF 2: Ser1 -Ile-Ser-Pro-Trp5 -Gly-Phe-Ser-Gly-Leu10 -Leu- Arg-Trp-Ser-Tyr15 ) selected from a random phage display library (RPL). A pep- tide-concentration-dependent formation of an RNA-peptide complex was clearly ob­servable by CEMSA. In the presence of the peptide the capillary electrophoresis (CE) peak for triply methylated ASL Phe shifted from 18.16 to 20.90 min. Formation of the complex was not observed when an unmethylated version of ASL Phe was used. The second system studied consisted of the (+18)-(+44) fragment of the trans-activation response element of human immunodeficiency virus type 1 (TAR RNA HIV-1) and a 9-amino-acid peptide of the trans-activator of transcription protein (Tat HIV-1) Tat(49–57)-NH2 (named Tat1: Arg49-Lys-Lys-Arg52-Arg-Gln-Arg-Arg- Arg57-NH2). In the presence of Tat(49–57)-NH2 a significant shift of migration time of TAR from 18.66 min to 20.12 min was observed. Methylation of a residue Arg52->Arg(Me)2, crucial for TAR binding, strongly disrupted formation of the complex. Only at a high micromolar peptide concentration a poorly shaped, broad peak of the complex was observed. CE was found to be an efficient and sensitive method for the analysis of methylation effects on interaction of biomolecules.

Słowa kluczowe

EN

methylated nucleoside; RNA-peptide interaction; arginine methylation; capillary electrophoresis; methylation

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 2003
• Tom: 50
• Numer: 3
• Opis fizyczny: p.857-864,fig.

Twórcy

autor

Mucha P.

• University of Gdansk, J.Sobieskiego 18, 80-952 Gdansk, Poland

autor

Szyk A.

autor

Rekowski P.

autor

Agris P.F.

Bibliografia

• Agris PF. (1996) The importance of being modified: roles of modified nucleosides and Mg2+ in RNA structure and function. Prog Nucleic Acid Res Mol Biol. ; 53: 79-129.
• Aletta JM, Cimato TR, Ettinger MJ. (1998) Protein methylation: a signal event in post-translational modification. Trends Biochem Sci.; 23: 89-91.
• Ashraf SS, Sochacka E, Cain R, Guenther R, Malkiewicz A, Agris PF. (1999) Single atom modification (O-->S) of tRNA confers ribosome binding. RNA.; 5: 188-94.
• Auld DS, Schimmel P. (1995) Switching recognition of two tRNA synthetases with an amino acid swap in a designed peptide. Science.; 267: 1994-6.
• Calnan BJ, Tidor B, Biancalana S, Hudson D, Frankel AD. (1991) Arginine-mediated RNA recognition: the arginine fork. Science.; 252: 1167-71.
• Causse E, Siri N, Arnal JF, Bayle C, Malatray P, Valdiguie P, Salvayre R, Couderc F. (2000) Determination of asymmetrical dimethylarginine by capillary electrophoresis-laser-induced fluorescence. J Chromatogr B Biomed Sci Appl.; 741: 77-83.
• Gary JD, Clarke S. (1998) RNA and protein interactions modulated by protein arginine methylation. Prog Nucleic Acid Res Mol Biol.; 61: 65-131.
• Heegaard NH, Kennedy RT. (1999) Identification, quantitation and characterization of biomolecules by capillary electrophoretic analysis of binding interactions. Electrophoresis.; 20: 3122-33.
• Helm M, Giege R, Florentz C. (1999) A Watson-Crick base-pair-disrupting methyl group (m1A9) is sufficient for cloverleaf folding of human mitochondrial tRNALYs. Biochemistry.; 38: 13338-46.
• Karn J. (1999) Tackling Tat. J Mol Biol.; 293: 235-54.
• Liebich HM, Xu G, Di Stefano C, Lehmann R. (1998) Capillary electrophoresis of urinary normal and modified nucleosides of cancer patients. J Chromatogr A.; 793: 341-7.
• Lischwe MA, Roberts KD, Yeoman LC, & Busch H (1982) Nucleolar specific acidic phosphoprotein C23 is highly methylated. J Biol Chem.; 257: 14600-2.
• Mucha P, Szyk A, Rekowski P, Weiss PA, Agris PF. (2001) Anticodon domain methylated nucleosides of yeast tRNA(Phe) are significant recognition determinants in the binding of a phage display selected peptide. Biochemistry.; 40: 14191-9.
• Mucha P, Szyk A, Rekowski P, Barciszewski J. (2002) Structural requirements for conserved Arg52 residue for interaction of the human immunodeficiency virus type 1 trans-activation responsive element with trans-activator of transcription protein (49-57) Capillary electrophoresis mobility shift assay. J Chromatogr A.; 968: 211-20.
• Muramatsu T, Nishikawa K, Nemoto F, Kuchino Y, Nishimura S, Miyazawa T, Yokoyama S. (1988) Codon and amino- acid specificities of a transfer RNA are both converted by a single post-transcriptional modification. Nature.; 336: 179-81.
• Putz J, Florentz C, Benseler F, Giege R. (1994) A single methyl group prevents the mischarging of a tRNA. Nat Struct Biol; 1: 580-2.
• Todorov TI, Morris MD. (2002) Comparison of RNA single-stranded DNA and double-stranded DNA behavior during capillary electrophoresis in semidilute polymer solutions. Electrophoresis.; 23: 1033-44.
• Yarian C, Marszalek M, Sochacka E, Malkiewicz A, Guenther R, Miskiewicz A, Agris PF. (2000) Modified nucleoside dependent Watson-Crick and wobble codon binding by tRNALys species. Biochemistry.; 39: 13390-5.
• Yeung ES. (1999) Study of single cells by using capillary electrophoresis and native fluorescence detection. J Chromatogr A.; 830: 243-62.