Figure 5.18: R2D2 dsRBD binding
A: Binding curves of R2D2 dsRBD1 and dsRBD2 to siRNA. Binding curves were generated as described. B: KD values for dsRBD binding to five different RNA substrates. KD values of one protein - RNA pair were obtained by fitting each Anisotropy binding curve separately with the Hill formula (equation 4.1) and averaging the obtained values. The indicated errors rep-resent the standard deviation of these values. The dsRBDs do not seem to exhibit substrate specificity.
more weakly than Loqs dsRBDs. The missing conservation of AA residues involved in RNA binding supports the latter. Like Loqs dsRBDs, R2D2 dsRBDs do not seem to preferentially bind a particular RNA substrate.
5.5 Interplay of the two Loquacious double-stranded RNA
Figure 5.19: Full length LoqsPD and Loqs DeltaNC show enhanced RNA binding affinity A: Binding curves of all Loqs constructs to siRNA. Binding curves were generated as de-scribed. LoqsPD full length and Loqs DeltaNC - siRNA binding curves are shifted to lower protein concentrations compared to those of the dsRBDs, indicating a lower KD. B: KDvalues for siRNA binding by all the Loqs constructs. KDvalues of protein - siRNA pairs were obtained as described. The double domain constructs bind up to five times stronger than the individual dsRBDs. C: EMSA experiments with all Loqs constructs and siRNA. EMSAs were conducted as described. The lanes are arranged in a way that the added protein amount is the same for lanes at the same position. EMSAs confirmed the tighter siRNA binding of the double domain proteins compared to the single domains.
Figure 5.20: Protein constructs used in binding experiments
A: Schematic representation of LoqsPD and R2D2. dsRBDs are depicted as blue squares.
The amino acids constituting the respective protein constructs are depicted below. B: Purified protein constructs. The respective molecular masses are: LoqsPD dsRBD1: 9kD, dsRBD2:
9.1kD, DeltaNC: 21kD; R2D2 dsRBD1: 7.1kD, dsRBD2: 7.8kD, DeltaNC: 17.4kD. Purification of R2D2 DeltaNC always resulted in the double band seen here.
5.5.2 Full length LoqsPD has an inherent bias towards siRNA binding
The commitment of LoqsPD to the siRNA biogenesis pathway might arise from substrate specificity of LoqsPD, from interaction of LoqsPD with Dcr-2 or a combination of both. In anisotropy measurements, the individual dsRBDs did not show any substrate specificity, there-fore the interplay of both domains might be necessary for substrate distinction. Since Loqs DeltaNC constitutes the core of LoqsPB as well as LoqsPD, the PD specific parts might also be necessary to increase the affinity for siRNA compared to miRNA. To test this, I determined the binding affinities of Loqs DeltaNC and LoqsPD full length to the complete set of RNA substrates (figure 5.15). Loqs DeltaNC bound all RNA substrates with similar affinity, with a slight trend for stronger si and dsRNA binding (figure 5.21A), which was not significant (see Appendix 6.6). Full length LoqsPD showed a significantly increased affinity for siRNA and dsRNA compared to Loqs DeltaNC, whereas their affinity for the mismatched substrates was approximately the same. The preference for perfectly matched substrates can also be seen in EMSAs of full length LoqsPD with the various RNAs (figure 5.21B).
Figure 5.21: Full length LoqsPD has an inherent bias towards siRNA binding
A: KDvalues for Loqs DeltaNC and LoqsPD full length binding to five different RNA substrates.
KD values of one protein - RNA pair were obtained as described. LoqsPD full length exhibits significantly stronger binding of completely base paired RNA substrates of a sufficient length compared to mismatched RNA substrates and the 14nt dsRNA. B: EMSA experiments with LoqsPD full length and various RNA substrates. EMSAs were conducted as described. EM-SAs confirmed the tighter siRNA and dsRNA binding by LoqsPD full length compared with the other RNA substrates.
5.5.3 Full length LoqsPD has the highest propensity to distinguish between RNA substrates
As can already be inferred from its increased siRNA binding strength, the ability to discriminate between RNA substrates is most pronounced in full length LoqsPD, and the differences in binding strengths for si- and miRNA targets were significant in an unpaired heteroscedastic t-test (see Appendix 6.6). This can already be seen in the averaged binding curves of the four protein constructs, which run very close to each other for all the substrates, except those of full length LoqsPD, which separate to some extent (see figure 5.22A).
To visualize the different binding behavior to the various substrates, the KD values of each protein construct were normalized to their affinity for siRNA, and the change in affinity for sub-strates with different structure and length were visualized (figure 5.22B, left and right, resp.).
Only two dsRBDs together showed a preferential binding to the siRNA mimic compared to miRNA-like structures, and only the distinction made by full length LoqsPD is significant. This argues for involvement of residues beyond the dsRBDs in limiting the substrate range of Lo-qsPD. For no protein construct the affinity for the longer dsRNA is increased, whereas the affinity for the 14nt RNA is marginally decreased for all proteins. The affinity of LoqsPD full length for the 14nt RNA is decreased significantly compared with siRNA and dsRNA sub-strates, endorsing the idea of additional residues involved in binding, which the shorter oligo might not be able to accommodate. Nevertheless, the 14nt oligo is bound remarkably well by all Loqs constructs.
Figure 5.22: Full length LoqsPD has the highest propensity to distinguish between RNA substrates
A: Overview over binding curves of all Loqs constructs to all RNA substrates. Binding curves were generated as described. Compared to the dsRBD2 binding curves, the LoqsPD full length binding curves are more expanded, indicating more variability in the KDvalues. B: For better visualization of substrate specificity of the Loqs constructs, the KDs shown in the bar graphs of figure 5.16 and 5.21 were normalized to the KD of the respective siRNA binding of each protein construct. Left: Comparison of substrate preference based on the dsRNA structure. Only the double domain constructs show decreased binding affinity for mismatched dsRNA substrates, with LoqsPD full length making the largest difference. Right: Comparison of substrate preference based on dsRNA length. Length increase does not lead to an in-creased binding affinity of any substrate, Length decrease results in dein-creased binding affinity, particularly of LoqsPD full length.
5.5.4 LoqsPD binds two different sequences with similar affinity
To exclude any sequence specific effects, the binding affinity of LoqsPD full length to a siRNA and miRNA mimic based on the sequence of miR8 (figure 5.23A) was measured, which yielded KDvalues comparable to the ones determined for the bantam derived oligos (figure 5.23B). In addition, the difference between the siRNA and miRNA substrates was more pro-nounced than the difference between the two sequences, as can be seen in the corresponding binding curves (figure 5.23C).
Figure 5.23: LoqsPD full length binding to two different sequences
A: The two different RNA sequences tested for binding to LoqsPD full length. The bantam-derived sequences were used in the preceding binding experiments, the miR8-bantam-derived se-quences are introduced here. B: KDvalues for LoqsPD full length binding to the two different siRNA and miRNA substrates. KD values of one protein - RNA pair were obtained as de-scribed. C: Binding curves of LoqsPD full length to the two different siRNA and miRNA sub-strates. Binding curves were generated as described. KD values are approximately the same for both sequences, the differences between the substrates is bigger than the differences between the two sequences.
5.5.5 Comparison with R2D2 DeltaNC
I also measured binding of R2D2 DeltaNC to the RNA substrates. The purified protein always presented a double-band, which might indicate confined degradation. Even with 10µM protein added, no real plateau in the binding curve could be reached (figure 5.24). Either R2D2 alone is not able to bind siRNA at physiological concentrations (as postulated, [59]), or it is simply
Figure 5.24: R2D2 DeltaNC binding
Binding curve of R2D2 full length to the siRNA, dsRNA and miRNA substrate. Binding curves were generated as described. No plateau was reached for any of the substrates, indicating ei-ther a KDabove the range covered in the protein titration or unspecific protein-RNA absorption due to misfolded protein.
not stable enough to retain its proper fold without Dcr-2 and the increase in Anisotropy values is due to trace amounts of correctly folded protein or unspecific protein-RNA adsorption.