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Arginine methylation analysis of the splicing-associated SR protein SFRS9-SRP30C

100%

Bressan G. C., Moraes E. C., Monfiolli A. O., Kuniyoshi T. M., Passos D. O., Gomes M. D., Kobarg J.

Cellular and Molecular Biology Letters

2009

tom 14

nr 4

657-669

The human SFRS9/SRp30c belongs to the SR family of splicing regulators. Despite evidence that members of this protein family may be targeted by arginine methylation, this has yet to be experimentally addressed. In this study, we found that SFRS9 is a target for PRMT1-mediated arginine methylation in vitro, and that it is immunoprecipitated from HEK-293 lysates by antibodies that recognize both mono- and dimethylated arginines. We further observed that upon treatment with the methylation inhibitor Adox, the fluorescent EGFP-SFRS9 re-localizes to dot-like structures in the cell nucleus. In subsequent confocal analyses, we found that EGFP-SFRS9 localizes to nucleoli in Adox-treated cells. Our findings indicate the importance of arginine methylation for the subnuclear localization of SFRS9.

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

88%

Mucha P., Szyk A., Rekowski P., Agris P. F.

Acta Biochimica Polonica

2003

tom 50

nr 3

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.

Ograniczanie wyników

1 Acta Biochimica Polonica

1 Cellular and Molecular Biology Letters

1 Agris P. F.

1 Bressan G. C.

1 Gomes M. D.

1 Kobarg J.

1 Kuniyoshi T. M.

1 Monfiolli A. O.

1 Moraes E. C.

1 Mucha P.

1 Passos D. O.

1 Rekowski P.

1 Szyk A.

1 2009

1 2003

Wyniki wyszukiwania

Arginine methylation analysis of the splicing-associated SR protein SFRS9-SRP30C

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