Tài liệu Báo cáo khoa học: Identification of Ewing’s sarcoma protein as a G-quadruplex DNA- and

Identification of Ewing’s sarcoma protein as a

G-quadruplex DNA- and RNA-binding protein

Kentaro Takahama1,*, Katsuhito Kino2,*, Shigeki Arai3

, Riki Kurokawa3 and Takanori Oyoshi1

1 Department of Chemistry, Faculty of Science, Shizuoka University, Japan

2 Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Kagawa, Japan

3 Division of Gene Structure and Function, Saitama Medical University Research Center for Genomic Medicine, Japan

Introduction

The current knowledge of Ewing’s sarcoma (EWS)

derives primarily from studies of a group of dominant

oncogenes that arise due to chromosomal transloca￾tions in which EWS is fused to a variety of cellular

transcription factors [1–3]. EWS fusion proteins are

very potent transcription activators that depend on the

EWS N-terminal domain and a C-terminal DNA-bind￾ing domain contributed by the fusion partner [4–9].

EWS ⁄ATF1 is a potent constitutive activator of ATF￾dependent promoters [10]. The EWS N-terminal binds

directly to the RNA polymerase II subunit hsRPB7

and this interaction is thought to be important for

transactivation [11].

In contrast to EWS fusion proteins, however, the

normal function and the nucleic acid-binding proper￾ties of EWS remain poorly characterized. EWS belongs

to a family that includes the closely related proteins

translocated in liposarcoma and the TATA-binding

protein-associated factor 15 which are involved in

several aspects of gene expression [12–15]. This protein

family contains the transcriptional activation domain

in the N-terminal region and the RNA-binding domain

Keywords

Ewing’s sarcoma; G-quadruplex DNA;

G-quadruplex RNA; RGG motif; RNA-binding

protein

Correspondence

T. Oyoshi, Department of Chemistry,

Faculty of Science, Graduate School of

Science, Shizuoka University, 836 Oya,

Suruga, Shizuoka 422-8529, Japan

Fax: +81 54 237 3384

Tel: +81 54 238 4760

E-mail: stohyos@ipc.shizuoka.ac.jp

*These authors contributed equally to this

work

(Received 27 August 2010, revised 23

December 2010, accepted 13 January 2011)

doi:10.1111/j.1742-4658.2011.08020.x

The Ewing’s sarcoma (EWS) oncogene contains an N-terminal transcrip￾tion activation domain and a C-terminal RNA-binding domain. Although

the EWS activation domain is a potent transactivation domain that is

required for the oncogenic activity of several EWS fusion proteins, the nor￾mal role of intact EWS is poorly characterized because little is known

about its nucleic acid recognition specificity. Here we show that the

Arg-Gly-Gly (RGG) domain of the C-terminal in EWS binds to the G-rich

single-stranded DNA and RNA fold in the G-quadruplex structure.

Furthermore, inhibition of DNA polymerase on a template containing a

human telomere sequence in the presence of RGG occurs in an RGG

concentration-dependent manner by the formation of a stabilized G-quad￾ruplex DNA–RGG complex. In addition, mutated RGG containing Lys

residues replacing Arg residues at specific Arg-Gly-Gly sites and RGG con￾taining Arg methylated by protein arginine N-methyltransferase 3 decrease

the binding ability of EWS to G-quadruplex DNA and RNA. These find￾ings suggest that the RGG of EWS binds to G-quadruplex DNA and

RNA via the Arg residues in it.

Abbreviations

dsHtelo, human telomere duplex DNA; EAD, Ewing’s sarcoma activation domain; EMSA, electrophoretic mobility shift assay; ETS, external

transcribed spacer; EWS, Ewing’s sarcoma; FMRP, fragile X mental retardation protein; GST, glutathione S-transferase; Htelo, human

telomere DNA; mut Htelo, mutated human telomere; mut rHtelo, mutated human telomere RNA; PRMT3, protein arginine

N-methyltransferase 3; RBD, RNA-binding domain; rHtelo, human telomere RNA; RRM, RNA recognition motif; ZnF, zinc finger.

988 FEBS Journal 278 (2011) 988–998 ª 2011 The Authors Journal compilation ª 2011 FEBS