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RESEARCH (English)

G-quadruplex binding proteins

G-quadruplex structures are formed at specific G-rich regions in the genome, mRNA, and non-coding RNA. These structures comprise two or more stacked guanine-tetrad planes and a monovalent cation such as K+ or Na+. G-quadruplex DNA regulates transcription, telomere elongation, epigenetics, and replication. G-quadruplex RNA is thought to have biologic functions, such as transcriptional regulation, RNA processing, mRNA localization and translation regulation. G-quadruplex structures are folded or unfolded by their binding proteins and these systems might affect biologic functions of them. In order to study the function of G-quadruplexes, we have to identify G-quadruplex binding proteins. We have already found G-quadruplex-binding proteins, Translocated in liposarcoma/Fused in sarcoma (TLS/FUS) and Ewing’s sarcoma (EWS), which contain the RNA recognition motif (RRM) and Arg-Gly-Gly (RGG) domains. The RGG domain is frequently found in G-quadruplex binding proteins. Tyr, Phe and Arg in the RGG domain are important for the G-quadruplex binding of the RGG domain. A recent study suggested that the beta-turn of the RGG domain might be a key structure for recognizing the G-quadruplex.

DNA- and RNA-binding protein. Takahama, K., Kino, K., Arai, S., Kurokawa, R., Oyoshi, T. FEBS. J. 2011, 278, 988-998.

Loop lengths of G-quadruplex structures affect the G-quadruplex DNA binding selectivity of the RGG motif in Ewing’s sarcoma. Takahama, K., Sugimoto, C., Arai, S., Kurokawa, R., Oyoshi, T. Biochemistry 2011, 50, 5369-5378.

G-quadruplex-proximity protein labeling based on peroxidase activity. Masuzawa, T., Sato, S., Niwa, T., Taguchi, H., Nakamura, H., Oyoshi, T. Chem. Commun. 2020, 56, 11641-11644.

 

Functions of G-quadruplexes

TLS/FUS binds to telomere DNA and telomeric repeat-containing RNA (TERRA) with G-quadruplex structure specificity and together they form a ternary complex. Moreover, TLS/FUS directly interacts with Suv4-20h2 and TERRA to promote histone modifications with the histone H3 modifying enzyme. These interactions facilitate the trimethylation of histone H4 at lysine 20 (H4K20me3) and histone H3 at lysine 9 (H3K9me3), respectively, and act to maintain heterochromatin and control telomere length.

Regulation of Telomere Length by G-Quadruplex Telomere DNA- and TERRA-Binding Protein TLS/FUS. Takahama, K., Takada, A., Tada, S., Shimizu, M., Sayama, K., Kurokawa, R., Oyoshi, T. Chem. Biol. 2013, 20, 341-350. 

 

G-quadruplex binding ability of RGG domain

The Arg-Gly-Gly repeat (RGG) domain is an evolutionarily conserved sequence found in DNA- or RNA-binding proteins. We found that RGG domain in C-terminal region of TLS/FUS and EWS bound to G-quadruplex, however, it does not mean that all RGG domains in DNA- or RNA-binding proteins can bind to G-quadruplex. Some structural features are required for it.

1. Tandem Arg-Gly-Gly repeat

2. Existence of Phe and Tyr close to Arg-Gly-Gly repeat

3. beta-turn structure of Arg-Gly-Gly

G-quadruplex Binding Ability of TLS/FUS Depends on the β-Spiral Structure of the RGG Domain. Yagi, R., Miyazaki, T., Oyoshi, T. Nucleic Acids Res. 2018, 46, 5894-5901.

Roles of the RGG domain and RNA recognition motif of Nucleolin in G‑quadruplex stabilization. Masuzawa, T. and Oyoshi, T. ACS Omega 2020, 5, 5202-5208.

 

Development of G-quadruplex DNA and RNA binding peptide

Tyr- or Phe-containing polypeptide engineered from the RGG domain in TLS/FUS selectively bind G-quadruplex DNA and RNA, respectively. These molecules revealed the different role the G-quadruplex of DNA and RNA in histone modification at the telomere. We expect that these will be useful for elucidating G-quadruplex functions in living cells and might have potential pharmaceutical implications.

Specific binding of modified RGG domain in TLS/FUS to G-quadruplex RNA: Tyrosines in RGG domain recognize 2’-OH of the riboses of loops in G-quadruplex. Takahama, K., Oyoshi, T. J. Am. Chem. Soc. 2013, 135, 18016-18019.

G-quadruplex DNA- and RNA-specific-binding proteins engineered from the RGG domain of TLS/FUS. Takahama, K., Miyawaki, A., Shitara, T., Mitsuya, K., Morikawa, M., Hagihara, M., Kino, K., Yamamoto, A., Oyoshi, T. ACS Chem. Biol. 2015, 10, 2564-2569.