Abstract
We examine the role of the heterogenous ribonucleoprotein U (hnRNP U) as a G-quadruplex binding protein in human cell lines. Hypothesizing that hnRNP U is associated with telomeres, we investigate what other telomere-related functions it may have. Telomeric G-quadruplexes have been fully characterized in vitro, but until now no clear evidence of their function or in vivo interactions with proteins has been revealed in mammalian cells. Techniques used were immunoprecipitation, DNA pull-down, binding assay, and Western blots. We identified hnRNP U as a G-quadruplex binding protein. Immunoprecipitations disclosed that endogenous hnRNP U associates with telomeres, and DNA pull-downs showed that the hnRNP U C-terminus specifically binds telomeric G-quadruplexes. We have compared the effect of telomere repeat containing RNA (TERRA) on binding between hnRNP U and telomeric (Tel) or single- stranded Tel (ssTel) oligonucleotides and found that ssTel binds stronger to TERRA than to Tel. We also show that hnRNP U prevents replication protein A (RPA) accumulation at telomeres, and the recognition of telomeric ends by hnRNP suggests that a G-quadruplex promoting protein regulates its accessibility. Thus, hnRNP U-mediated formation has important functions for telomere biology.
Highlights
Telomeres are nucleoprotein complexes that constitute the natural ends of eukaryotic chromosomes.One strand is G-rich and forms a single-stranded 30 overhang, whereas the complementary strand isC-rich and has a recessed 50 end [1]
We find that Heterogenous ribonucleoprotein U (hnRNP U) prevents replication protein A (RPA) from recognizing telomeric ends, and that hnRNP
Efficiently than that between Flag-hnRNP U and single- stranded Tel (ssTel). These results suggested that telomere repeat containing RNA (TERRA) competes for binding, Cells 2019, 8, x but the preferred substrate is telomeric DNA
Summary
Telomeres are nucleoprotein complexes that constitute the natural ends of eukaryotic chromosomes. C-rich and has a recessed 50 end [1]. The essential role of telomeres is to protect chromosome ends from recombination and fusion and from being recognized as broken DNA ends. Heterogenous ribonucleoprotein U (hnRNP U) has been reported to be part of a telomerase complex, but its functions in telomeres has not been precisely known, necessitating further studies. HnRNP U has been suggested to be a telomere-end binding protein, contributing to telomere length regulation [2]. The length of the telomeric DNA gradually shortens as cells divide
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