Mammalian telomeres comprise noncoding TTAGGG repeats in double-stranded regions with a single-stranded TTAGGG repeat 3' overhang and are bound by a multiprotein complex with a telomeric repeat-containing RNA (TERRA) containing a UUAGGG repeat as a G-quadruplex noncoding RNA. TLS/FUS is a human telomere-binding protein that was first identified as an oncogenic fusion protein in human myxoid and round-cell liposarcoma. Here, we show that the Arg-Gly-Gly domain in the C-terminal region of TLS forms a ternary complex with human telomere G-quadruplex DNA and TERRA in vitro. Furthermore, TLS binds to G-quadruplex telomere DNA in double-stranded regions and to G-quadruplex TERRA, which regulates histone modifications of telomeres and telomere length in vivo. Our findings suggest that the G-quadruplex functions as a scaffold for the telomere-binding protein, TLS, to regulate telomere length by histone modifications.

The Ewing’s sarcoma (EWS) oncogene contains an N‐terminal transcription 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 normal 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‐quadruplex DNA–RGG complex. In addition, mutated RGG containing Lys residues replacing Arg residues at specific Arg‐Gly‐Gly sites and RGG containing Arg methylated by protein arginine N‐methyltransferase 3 decrease the binding ability of EWS to G‐quadruplex DNA and RNA. These findings suggest that the RGG of EWS binds to G‐quadruplex DNA and RNA via the Arg residues in it.

Telomeric repeat-containing RNA (TERRA), which contains tandem arrays of short RNA repeats, r(UUAGGG), is an integral component of the telomere and contributes to telomeric heterochromatin formation and telomere-length regulation. TERRA forms a G-quadruplex, but the biologic significance of its G-quadruplex formation is unknown. Compounds that selectively bind to G-quadruplex RNA are useful for understanding G-quadruplex TERRA. Here we report that an engineered RGG domain translocated in liposarcoma (TLS) specifically binds to G-quadruplex TERRA. The Arg-Gly-Gly repeat (RGG) TLS binds to G-quadruplex human telomere DNA and TERRA simultaneously, but we show that substitution of Tyr for Phe in the RGG domain of TLS (TLSRGG3Y) converts its binding specificity solely toward G-quadruplex TERRA. TLSRGG3Y binds to dG tetrads with abasic RNA loops, but fails to bind to rG tetrads without loops or dG tetrads with abasic DNA loops. These findings suggest that TLSRGG3Y binds to loops within the G-quadruplexes of TERRA by recognizing the 2'-OH of the riboses. To our knowledge, TLSRGG3Y is the first known molecule that specifically recognizes the 2'-OH of the riboses of loops in the G-quadruplex. TLSRGG3Y will be useful for investigating the role of the G-quadruplex form of TERRA without affecting G-quadruplex telomere DNA functions.

The G-quadruplex nucleic acid structural motif is a target for designing molecules with potential anticancer properties. To achieve therapeutic selectivity by targeting the G-quadruplex, the molecules must be able to differentiate between the DNA of different G-quadruplexes. We recently reported that the Arg-Gly-Gly repeat (RGG) of the C-terminus in Ewing's sarcoma protein (EWS), which is a group of dominant oncogenes that arise due to chromosomal translocations, is capable of binding to G-quadruplex telomere DNA and RNA via arginine residues and stabilize the G-quadruplex DNA form in vitro. Here, we show that the RGG of EWS binds preferentially to G-quadruplexes with longer loops, which is not related to the topology of the G-quadruplex structure. Moreover, the G-quadruplex DNA binding of the RGG in EWS depends on the phosphate backbone of the loops in the G-quadruplex DNA. We also investigated the G-quadruplex DNA binding activity of the N- and C-terminally truncated RGG to assess the role of the regions in the RGG in G-quadruplex DNA binding. Our findings indicate that the RGG and the other arginine-rich motif of residues 617-656 of the RGG in EWS are important for the specific binding to G-quadruplex DNA. These findings will contribute to the development of molecules that selectively target different G-quadruplex DNA.

Human telomere DNA (Htelo) and telomeric repeat-containing RNA (TERRA) are integral telomere components containing the short DNA repeats d(TTAGGG) and RNA repeats r(UUAGGG), respectively. Htelo and TERRA form G-quadruplexes, but the biological significance of their G-quadruplex formation in telomeres is unknown. Compounds that selectively bind G-quadruplex DNA and RNA are useful for understanding the functions of each G-quadruplex. Here we report that engineered Arg-Gly-Gly repeat (RGG) domains of translocated in liposarcoma containing only Phe (RGGF) and Tyr (RGGY) specifically bind and stabilize the G-quadruplexes of Htelo and TERRA, respectively. Moreover, RGGF inhibits trimethylation of both histone H4 at lysine 20 and histone H3 at lysine 9 at telomeres, while RGGY inhibits only H3 trimethylation in living cells. These findings indicate that G-quadruplexes of Htelo and TERRA have distinct functions in telomere histone methylation.