A U-Tetrad Stabilizes Human Telomeric RNA G-Quadruplex Structure

Abstract

Telomeric repeat-containing RNA is a new noncoding RNA molecule recently discovered in mammalian cells. Here we report the structural features of human telomere RNA r(UAGGGU) in the presence of K(+) and Na(+). We demonstrated for the first time that a novel U-tetrad is formed at the 3' end of a parallel human telomeric RNA G-quadruplex. The U-tetrad dramatically stabilizes human telomeric RNA G-quadruplex structure, leading to an increase in melting temperature (T(m)) of 29 degrees C. The U-tetrad-stabilized telomeric RNA G-quadruplex structure adds considerably to our understanding of the diversity of RNA G-quadruplex architectures. It shows that the structure of base "quartets" is important in RNA assembly. The structural information will be invaluable for understanding the function of human telomere RNA.

Distinctive structural motifs of RNA G-quadruplexes composed of AGG, CGG and UGG trinucleotide repeats

“…For example, a small negative band at ∼290 nm in the CD spectrum of p(UGG) 2 U was attributed to the U-tetrad formation. A similar negative band had also been observed, but not discussed, in the CD spectra of other RNA sequences containing U-tetrads (55,59). …”

“…When the RNA concentration was increased, signals corresponding to the duplex diminished notably, and the G-quadruplex emerged as the dominant form (Figure 5Ad). The observed down-field shift of G:C imino resonance relative to that in the presence of K + could be related to the deshielding effect of the ion, or could reflect different metal binding sites within the G:C:G:C tetrad (55). In the ammonium buffer, only two sharp imino resonances at 12.63 and 12.71 ppm were visible and no signals corresponding to the G-quadruplex form could be determined (Figure 5Ac).…”

“…To verify whether the uridine residues constitute part of the G-quadruplex architecture, we assigned all guanosine and uridine imino protons based on the analysis of a 2D NOESY spectrum (data not shown). Each of the three observed uridine imino resonances exhibited a strong NOE to H5 and much weaker one to H6 protons (Supplementary Figure S11), a pattern that is distinctive of a U-tetrad motif (55). The 3′-end uridine imino proton at 11.08 ppm and that at 9.85 ppm, assigned to the internal uridine residue, sharpened with increasing temperature and were still observed at a high temperature, revealing the formation of two stable U-tetrads.…”

The 5′ Guanosine Tracts of Human Telomerase RNA Are Recognized by the G-Quadruplex Binding Domain of the RNA Helicase DHX36 and Function To Increase RNA Accumulation

“…Synthetic RNAs harboring on May 9, 2018 by guest http://mcb.asm.org/ hTR sequences 5Ј of the template can fold as a G-quadruplex (19,26). Importantly, unlike DNA, RNA uniformly adopts the parallel-strand orientation of quadruplex in vitro and in vivo (4,8,30,41). DHX36 has the ability to bind and resolve a model intermolecular G-quadruplex RNA (11).…”

“…Helicase domain proteins participate broadly in RNA processing, RNP biogenesis, and RNP regulation with strand-separating, strand-annealing, and RNP-remodeling activities potentially relevant to telomerase RNP maturation and function (24). DHX36 in particular has several potentially relevant substrates, because model oligonucleotides with the sequence of human telomeric DNA, telomeric RNA, or the 5Ј region of hTR can form Hoogsteen-base-paired G-quadruplex structures in vitro (4,8,19,26,30,41). We therefore investigated whether DHX36 associates with human telomerase and, if so, whether the interaction has defined sequence requirements or a more chaperone-like general specificity.…”