Using capillary electrophoresis to study methylation effect on RNA-peptide interaction.
AbstractMethylation of RNA and proteins is one of a broad spectrum of post-transcriptional/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 (Cm(32)), 2'-O-methylguanine (Gm(34)) and 5-methylcytidine (m(5)C(40))) (ASL(Phe)-Cm(32),Gm(34),m(5)C(40)) and a 15-amino-acid peptide (named t(F)2: Ser(1)-Ile-Ser-Pro-Trp(5)-Gly-Phe-Ser-Gly-Leu(10)-Leu- Arg-Trp-Ser-Tyr(15)) selected from a random phage display library (RPL). A peptide-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)-NH(2) (named Tat1: Arg(49)-Lys-Lys-Arg(52)-Arg-Gln-Arg-Arg- Arg(57)-NH(2)). In the presence of Tat(49-57)-NH(2) a significant shift of migration time of TAR from 18.66 min to 20.12 min was observed. Methylation of a residue Arg(52)-->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.
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