Interaction between LS2 RRM domains and G-quadruplex RNA

Biochemical and structural studies reported on various RRM domains have shown its preference for single-stranded nucleic acid form (Afroz, Cienikova et al. 2015). The RRM domain utilizes its β-sheet surface for RNA recognition, but the mode of binding is not always same. In the case of canonical RRM domains, two aromatic residues located on RNP1 site (β3-strand) and 1 aromatic residue on RNP2 site (β1-(β3-strand) are involved in the base stacking interactions with RNA nucleotides. Additional contacts are made by positively charged side chain at RNP1 site.

In the light of G-quadruplex formation by LS2 target RNA sequences, it was tempting to assume that LS2 RRMs interact with a single-stranded form of RNA as reported for hnRNP F RRMs (Samatanga, Dominguez et al. 2013). However, the NMR titrations of single RRM domains, linker-RRM2 as well as RRM1,2 with various guanosine-rich RNA ligands presented, show that LS2 RRM domains interact with a G-quadruplex form of RNA. RRM1 interacts with all the guanosine-rich RNA oligonucleotides used in the study, irrespective of their homogeneous conformation, whereas, RRM2 shows specificity for the 21mer-specific uniform G-quadruplex conformation (Figure 3.40, Figure 3.42). Therefore, this is the first study, which reports novel RRM-G-quadruplex interaction.

It is intriguing that for interaction with G-quadruplex RNA, LS2 RRM2 uses canonical mode of binding, as in presence of RNA, β-1 and β-3 strands (containing RNP2 and RNP1 sites respectively) shows maximum chemical shift changes and line broadening (Figure 3.37). In addition to RNP sites, residues located on other β-strands also show an effect in presence of

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RNA. On the other hand, in the case of LS2 RRM1, chemical shift perturbation analysis shows that residues located on the β-5 as well as C-terminal flexible part of LS2 RRM1 show maximum chemical shift changes (Figure 3.36). Chemical shift changes were also observed in the other parts of the protein, except α-2 helix and β-3 site harboring canonical RNP1 residues. This indicates that this RRM-RNA interaction is non-canonical, as RNP1 residues are not involved in RNA binding. The similar mode of binding is also reported for PTB RRM domains and Py tract interaction, in which β-3 strand doesn’t participate in the RNA Binding instead there is additional β-5 strand which is involved in RNA binding (Oberstrass, Auweter et al. 2005). But, in contrast, in the case of LS2 RRM1, there is apparently no additional β-strand observed (Figure 3.19).

It was intriguing to see whether, in the presence of the RRM1-RRM2 linker, RRM2 can interact with the 21mer RNA, as this linker weakly interacts with the β2 and β3-strands in RRM2, which are also involved in RNA binding. However, similar to RRM2, linker-RRM2 showed interaction with 21mer, characterized by line broadening of both RNP sites and additional β-strands (Figure 3.44). A region with helical propensity also showed chemical shift perturbations as well as line broadening. It would be important to understand the contribution of the linker residues in RNA binding. It could be speculated that linker-RRM2 has potential to bind RNA more tightly (because of additional RNA binding patch) than RRM2. On the other hand, the interaction of the linker with the β-strands of the RRM2 might provide additional specificity.

ITC experiments of RRM2 and linker-RRM2 with various RNA ligands should be performed to gain more insights about this.

NMR titration of linker-RRM2 with 21mer multiple conformations shows that protein does not bind to G-quadruplex RNA in multiple conformations. Whereas, upon a change in RNA conformation from multiple to uniform obtained through slow cooling, the protein-RNA binding was observed (Figure 3.46). This data again highlight that LS2 (in this case linker-RRM2) indeed shows specificity for G-quadruplex uniform conformation adopted by 21mer.

Similarly, the addition of G-quadruplex-specific inhibitor TMPyP4, results into disruption of linker-RRM2-21mer complex, indicating that RNA is G-quadruplex form in the RNA and is not unfolded into single-stranded form upon protein binding (Figure 3.47).

Although the dimeric G-quadruplex topology of the 21mer RNA is intriguing, it is possible that the natural LS2 ligands may not necessarily be dimeric. The SELEX pattern obtained for LS2 target RNA sequences rather hints that they may adopt intramolecular G-quadruplex. The physiological relevance of our findings is being probed by performing in vivo splicing assays.

For this purpose, the study further identifies possible mutants of LS2 RRM1,2 with retarded RNA-binding contribution from each RRM and linker respectively. These mutants will be used to perform in vivo splicing assays to validate the role of each RNA-binding element. The importance of G-quadruplex during in vivo splicing assays could be analyzed by using TMPyP4 to inhibit G-quadruplex formation as the intake inside the cell can be easily achieved.

Structural insights of RRM-G-quadruplex interaction will be important to understand this novel interaction. But, the aggregation-prone behavior of RRM1 and line-broadening of resonances observed for RRM2 and G-quadruplex interaction, poses difficulties in structural studies by NMR. It is possible that LS2 RRM domains bind to RNA in multiple registers, which

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results in the line broadening of the resonances. Given the symmetry of G-quadruplex, it could also be possible that two or more molecules of RRM2 interact with each RNA molecule, which could increase the molecular mass of the complex. As larger complexes tumble more slowly in the solution, resulting in a shortened transverse relaxation time, thereby causing line broadening. Hence, the line broadening observed for both LS2 and 21mer RNA in bound form could be attributed to increased molecular weight. SLS data did not provide any information about the stoichiometry of the RRM2-G-quadruplex complex since the complex separated while passing through the column, indicating that the affinity of the protein-RNA complex may not be strong enough (Figure 3.43). With the lack of information about stoichiometry of the complex formation, as well as the susceptibility of G-quadruplex conformation for salt type and concentration, crystallization trials for the RRM-G-quadruplex complex may not be successful.

Although a number of G-quadruplex structures have been solved so far using X-ray crystallography and NMR, information regarding how proteins interact with G-quadruplex structures remains elusive. Recently, solution NMR structure (Phan, Kuryavyi et al. 2011) and crystal structure (Vasilyev, Polonskaia et al. 2015) are reported for RGG peptide of FMRP protein and RNA duplex-quadruplex junction. Both structures suggest that shape complementarity and hydrogen bonding interactions play a crucial role in the interaction between RGG peptide and duplex-quadruplex junction. The data reveals that two Arginine side chains form crucial hydrogen bonds with the RNA duplex residues, whereas no apparent interaction was detected between peptide and G-quadruplex.

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Figure 4.2 Structures of G-quadruplex bound peptides. A. A reported solution structure of RGG peptide-sc 1 RNA, showing the interaction between RGG peptide with G-quadruplex and G-quadruplex-duplex junction. The peptide is colored red with important Arginine side chains in green, the duplex, junction and quadruplex regions are represented by magenta, orange, and cyan, respectively whereas sugar-phosphate backbone is in silver color. [Figure adapted from (Phan, Kuryavyi et al. 2011)]. B. Side surface representation of solution structure of the Rhau18–T95-2T complex. The intermolecular interactions between peptide residues and DNA G-quadruplex are shown. [Figure adapted from (Heddi, Cheong et al. 2015)].

On the other hand, solution NMR structure of N-terminal RHAU region and G-DNA quadruplex shows that peptide covers the top most G-quartet plane (Heddi, Cheong et al. 2015).

Positively charged Lysine and Arginine side chains mediate electrostatic interactions with negatively charged RNA phosphate groups. This interaction is similar to that of most G-quadruplex-specific ligands targeted for G-quadruplex binding.