Structure specific recognition of telomeric repeats containing RNA by the RGG-box of hnRNPA1

Abstract The telomere repeats containing RNA (TERRA) is transcribed from the C-rich strand of telomere DNA and comprises of UUAGGG nucleotides repeats in humans. The TERRA RNA repeats can exist in single stranded, RNA-DNA hybrid and G-quadruplex forms in the cell. Interaction of TERRA RNA with hnRNPA1 has been proposed to play critical roles in maintenance of telomere DNA. hnRNPA1 contains an N-terminal UP1 domain followed by an RGG-box containing C-terminal region. RGG-motifs are emerging as key protein motifs that recognize the higher order nucleic acid structures as well as are known to promote liquid-liquid phase separation of proteins. In this study, we have shown that the RGG-box of hnRNPA1 specifically recognizes the TERRA RNA G-quadruplexes that have loops in their topology, whereas it does not interact with the single-stranded RNA. Our results show that the N-terminal UP1 domain in the presence of the RGG-box destabilizes the loop containing TERRA RNA G-quadruplex efficiently compared to the RNA G-quadruplex that lacks loops, suggesting that unfolding of G-quadruplex structures by UP1 is structure dependent. Furthermore, we have compared the telomere DNA and TERRA RNA G-quadruplex binding by the RGG-box of hnRNPA1 and discussed its implications in telomere DNA maintenance.

* To whom correspondence should be addressed Email: singh@iisc.ac.in

Supplementary text
Figures S1-S5 Table S1 References for SI

Supplementary text
Characterization of TERRA RNA G-quadruplexes Table S1 shows the RNA sequences that were used in this study. Although these RNA sequences have been studied previously (1, 2), we deemed it necessary to characterize them under the current experimental conditions. Two RNA sequences were used as single stranded RNA control: an 18mer control RNA sequence (ssRNA-18) and a 24mer single stranded mutated TERRA sequence where two Gs are substituted with Cs (ssTERRA-mut) (Table S1).
These two RNA sequences are designed so that they do not form G-quadruplex and remain in the single stranded form in solution. 6mer TERRA-6, 12mer TERRA-12, and 24mer TERRA-24 RNA sequences are designed to form tetrameric, dimeric, and monomeric intramolecular RNA G-quadruplexes of parallel topologies respectively (Table S1). An abasic loop containing TERRA-24 sequence (abasicloopsTERRA-24) was devoid of bases in the loop residues of the RNA, was also used in this study (Table S1).
The CD spectra of the G-quadruplexes formed by the TERRA-6, TERRA-12 and TERRA-24 RNA sequences were acquired in physiologically relevant 100 mM KCl containing buffer (3).
TERRA-6 sequence forms a tetrameric, parallel G-quadruplex without loops in its structure.
TERRA-12 sequence adopts a dimeric, parallel G-quadruplex structure with two loops and TERRA-24 sequence adopts a monomeric, intramolecular, parallel G-quadruplex structure with three loops (1, 2). All the three RNA sequences form G-quadruplexes of parallel conformation that is characterized by positive maxima at 262 nm and minima at 240 nm in the CD spectra ( Fig. S1A). Further, the presence of peaks in the imino region of 1D 1 H NMR spectra confirms the formation of Hoogsteen hydrogen bonded G-quadruplex structures by these RNA sequences (Fig. S1B). The CD spectra of ssRNA-18 and ssTERRA-mut sequences corresponded to that of single-stranded RNA. This was further confirmed by recording the 1D 1 H NMR spectra that showed absence of hydrogen bonded imino peaks in the spectra (Fig. S1B).
The thermodynamic stabilities of TERRA-6, TERRA-12, and TERRA-24 RNA G-quadruplexes were monitored using CD thermal melting experiments in the presence of 100 mM KCl. The CD signal at 262 nm was followed as a function of increasing temperature. In all the cases, we observed two state, sigmoidal melting transitions. The T m of the intramolecular G-quadruplex, TERRA-24 was calculated to be 80.5°C (Fig. S1C). In a previous study, a T m of 64.6°C in the presence of 10 mM KCl had been reported for this sequence (1, 2). This confirms the influence of potassium concentration on the thermal stability of the RNA G- quadruplex (1, 4). Under the 3 same condition, dimeric TERRA-12 G-quadruplex exhibited a T m of 63.4°C, whereas the tetrameric TERRA-6 G-quadruplex exhibited a T m of 72.5°C. These results show that in the presence of 100 mM KCl, intramolecular TERRA-24 forms thermally most stable G-quadruplex structure followed by TERRA-6 and TERRA-12 structures respectively.
The formation of the RNA G-quadruplex structure by TERRA-24 in the presence of 20% PEG 200 was confirmed using CD spectroscopy (Fig. S2A). Formation of an intramolecular, pralallel G-quadruplex by the abasicloopsTERRA-24 RNA sequence was confirmed by CD spectroscopy (Fig. S2B) and 1D 1 H NMR spectroscopy (Fig. S2C).

ITC titration of TERRA-6 RNA with UP1+RGG
The interaction of UP1+RGG with RNA G-quadruplex was investigated using ITC. However no significant heat change was observed that suggests no interaction between UP1+RGG and TERRA-6 as probed using ITC (Fig. S4). Figure S1.