Zf1 specifically recognizes a GG motif

To study the RNA sequence specificities of Zf1, two different RNA sequences were titrated to the protein and the ¹H,¹⁵N-HSQC spectra were recorded subsequently (Figure 51).

Negligible chemical shift perturbations were observed upon titration of a C/U rich RNA (Figure 51, spectrum in pink) into the protein. Contrastingly, huge chemical shift changes were observed upon titration of an RNA containing an additional GG motif (as seen in the CSP plot).

The titration indicated that the formation of the complex was in intermediate-fast exchange on the chemical shift timescale, whereby some signals shifted in position with each titration point while some others simply disappeared. This clearly indicates that the Zf1 protein specifically recognizes a GG motif and does not bind to a C/U rich RNA.

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Figure 51 Zf1 requires a GG dinucleotide motif for RNA binding

(A) Superposition of ¹H,¹⁵N-HSQC spectra of the free Zf1 domain, bound to GGCU_7 (5’-CUUGGCU-3’) and CU_9 (5’-UCUCUUCUC-3’) RNA is shown in green, purple and pink, respectively. Zoom-ins of three residues are shown on the right. (B) The chemical shift perturbation plot clearly demonstrates that the chemical shifts between the free and CU rich RNA bound spectra are minimal while the Zf1 domain readily recognizes the GG motif containing RNA.

This is not surprising as it was previously suggested that RanBP2-type Zinc fingers preferentially bind to a consensus sequence AGGUAA (Nguyen, Mansfield et al. 2011). By varying bases at the 2^nd and 4^th position in the RNA sequence and measuring the binding affinity using fluorescence anisotropy titrations, the authors showed that RBM5 Zf1has the highest affinity (~250 nM) for AGGGAA and it has a 1.5-, 2- and 8-fold preference for guanine over uracil, adenine and cytosine at the 4^th position, respectively.

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Figure 52 Zf1 does not contribute towards binding to C/U rich RNA

(A) A superposition of ¹H,¹⁵N-HSQC spectra of RRM1-Zf1 free, Zf1 free and RRM1-Zf1 bound to C/U rich is shown in black, green and pink, respectively. Zoom-ins of two Zf1 residues show that their peak positions in C/U rich RNA-bound RRM1-Zf1 shift towards their respective positions in Zf1 alone. (B) ITC binding isotherms of RRM1 and RRM1-Zf1 with C/U rich RNA are shown. Upon addition of Zf1 to RRM1 in the tandem construct, there is not much gain in affinity as would be expected if both the domains contribute to RNA binding, comparing left and right panels.

In order to understand what happens in the two-domain context (RRM1-Zf1), NMR titrations of the C/U rich RNA into the protein were performed. An overlay of the ¹⁵N-HSQC spectra of free Zf1 and free and RNA bound RRM1-Zf1 shows that there are chemical shift perturbations observed in Zf1 domain in the RNA-bound form of RRM1-Zf1 (Figure 52A).

Zoomed-in views for two distinct amide signals in the Zf1 domain show that the NMR signals in the RNA-bound form of Zf1 shift towards that of the free Zf1 protein. Since we already

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know that Zf1 does not bind to C/U rich RNA, the shifts in Zf1 must occur due to partial displacement of the Zf1 rather than direct RNA binding.

To further confirm this, ITC binding isotherms of RRM1 and RRM1-Zf1 for binding to C/U rich RNA (Figure 52B) were recorded. RRM1 has a binding affinity of ~20 μM while that of RRM1-Zf1 is ~6 μM. The gain in affinity by adding an additional domain (Zf1) should be much greater than a factor of 3. It is possible that some residueal binding of Zf1 to the RNA occurs which is reflected in the 3-fold gain in affinity, only due to restraints in the conformational space. Since, Zf1 is still atached to RRM1, it could be conformationally restricted possibly leading to some interactions. Therefore, this provides further proof that Zf1 domain does not bind to a C/U rich RNA.

Now with the knowledge of binding specificities of Zf1, I tested another RNA oligo which contains both C/U rich motif for RRM1 binding and GG motif for Zf1 binding named as GGCU_12 (5’-UGGCUCUUCUCU-3’). This RNA oligo is also derived from the Caspase-2 pre-mRNA intronic sequence, upstream of ln100 element. An NMR titration of the RNA into RRM1-Zf1 protein shows significant chemicals shift perturbations (Figure 53A). The complex formation takes place on intermediate-fast exchange timescale, whereby some amide signals shift in position while some others disappear. It is noteworthy, that almost all the RRM1 amide signals can be traced and the NMR signals broadened beyond detection mostly belong to the Zf1 domain. I also recorded an ITC binding isotherm of this RNA for binding to RRM1-Zf1 and obtained a binding affinity of ~84 nM which compared to RRM1 binding to C/U rich RNA with ~20 μM affinity, is a gain of 24-fold in affinity.

Taken together, we now know that an RNA containing C/U rich element and a GG motif can bind to both RRM1 and Zf1 domains with binding affinity in the nanomolar range.

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Figure 53 RRM1 binds to C/U rich RNA while Zf1 specifically recognizes ‘GG motif

(A) A superposition of ¹H,¹⁵N-HSQC spectra of RRM1-Zf1 in free and RNA bound form (GGCU_12) is shown in black and red, respectively. It is clear from the chemical shifts that both the domains bind to RNA (top zoom-in depicts RRM1 residues while the bottom one depicts Zf1 residues). (B) ITC binding isotherm of RRM1-Zf1 with RNA oligo containing both C/U rich and GG rich motifs (GGCU_12). The binding affinity of the tandem domain increases by an order of magnitude upon comparison with that of RRM1 alone (Figure 52B, left panel).

5.5.2. Probing residues important for RNA binding in RRM1-Zf1 using point mutations