rna polymerase ii br Acknowledgements This work was supporte

Acknowledgements This work was supported under the National Natural Science Foundation of China (Grant numbers 31200576, 21472197, 21675162), Beijing Natural Science Foundation (Grant No. 7182189), and Project supported by the Joint Funds of the National Natural Science Foundation of China (Grant No. U1432250).
Introduction G-quadruplex structure, a specific type of secondary structure formed by DNA or RNA strands found in telomeres, promoters, exons, and UTR regions, have been proven to play an important role in replication and translation processes [1]. Recently, G-quadruplexes have also been researched as a potential target for cancer treatment [2]. New small molecules have been developed to selectively recognize and bind G-quadruplexes to modulate gene rna polymerase ii [[3], [4], [5], [6]]. MicroRNAs (miRNAs) were naturally occurring, highly conserved, and short noncoding RNA molecules. They were 17–27 nucleotides long and normally regulated gene expression at the post-transcriptional level by binding to complementary 3′UTR in the mRNA sequence, which in turn led to translational inhibition and gene silencing. One miRNA might regulate the functions of multiple mRNAs, and these mRNAs were usually associated with the same disease or occurred in the same signal transduction pathway [7]. The development of a miRNA inhibitor associated with a specific disease would allow the simultaneous regulation of multiple associated mRNAs targeting various signaling pathways and functions. Therefore, miRNA-targeted disease treatment has attracted growing research interests [8]. At present, several strategies for regulating mRNA function through miRNA targeting have been described. An exogenously introduced anti-miR could complementarily pair with an overexpressed miRNA and competed with the target gene mRNA, thereby inhibiting miRNA binding to the mRNA 3′UTR region [9], which might also lead to sequence-dependent [10] or non-sequence-specific off-target effects [11]. For these reasons, regulating miRNAs function with small molecules has attracted considerable research interests [12,13]. Very little research has focused on the relationship between miRNAs function and the G-quadruplex structure. Some studies have shown that G-quadruplexes produced during miRNA formation had an impact on the maturation process [14]. Besides, the G-rich sequence in the 3′UTR region of the FADS2 gene affected the binding of mRNA to miR-331-3p after the formation of the G-quadruplex structure [15]. Recently, by bioinformatics analysis, we found that 152 mature human miRNAs containing G-rich sequences such as miR-197-5p, miR-765, miR-1587 [16], miR-3620 [17], miR-4507 and miR-5196 (Table S1), had a high probability of forming intramolecular G-quadruplexes. Among them, miR-1587 contained regular four G-tracts and exhibited high expression in HeLa cells. Recent studies have also shown that Glioma-associated mesenchymal stem cells secreted miR-1587 by releasing exosomes, targeting and inducing downregulation of NCOR1 expression in Glioma stem cells and thereby promoting proliferation and colony formation and increasing cells tumorigenicity [18]. The results of this study highlighted the importance of miR-1587. Therefore, miR-1587 was selected as a model to test G-quadruplex formation in mature human miRNAs and targeted functional regulation by G-quadruplex structure and its ligands.
Experimental section
Results and discussion We performed ESI-MS, CD and NMR experiments to investigate the formation and properties of the miR-1587 G-quadruplex. In the presence of NH4OAc, the ESI-MS spectrum was dominated by a monomeric miR-1587 G-quadruplex (abbreviated to Q) peak with five negative charges and two adducted ammonium ions ([S + 2NH4+ − 7H+]5−, abbreviated to [Q]5−), which was confirmed by the isotopic distribution in a high resolution mass spectrum [16]. The CD spectrum showed that miR-1587 exhibited a maximum positive band near 260 nm and a minimum negative band near 240 nm in the presence of 150 mM KCl, indicating the formation of a parallel miR-1587 G-quadruplex structure. Meanwhile, the CD melting curve suggested the high thermo-stability in 150 mM KCl and a Tm value of 66 °C in 5 mM KCl, indicating the high thermo-stability of miR-1587 G-quadruplex. In the presence of potassium ions, miR-1587 displayed a group of proton peaks in 10.8–11.8 ppm in 1H NMR, which was a characteristic chemical shift region for imino protons of guanines participating in the formation of G-quartets (Fig. S1). The results of ESI-MS, CD and NMR spectra confirmed that miR-1587 folded into a stable parallel G-quadruplex structure with high thermo-stability. Next, we focused on the target gene identification and functional regulation of miR-1587.