Stress granule localization of hsPURA depends on its ability to bind RNA.

A Schematic overview of the hsPURA protein. Patient-derived mutations experimentally assessed in this study are marked in red.

B In HeLa cells stress granules were identified by G3BP1 immunostaining (magenta) and overexpressed hsPURA (yellow) by its N-terminal Flag-tag. Nuclei were stained with DAPI (blue). Representative stress granules are marked with red arrows in the zoomed-in area. Except for hsPURA m11, all overexpressed versions of hsPURA (wild-type and mutant) showed strong stress-granule localization.

C Box-Whiskers plot of quantified intensities of hsPURA staining in stress granules above cytoplasmic background levels upon arsenite-induced stress treatment (see A). Three biological replicates (indicated by different icons) with 66 stress granules each were quantified for each cell line. Box-Whiskers-Plot of average signal intensity per repeat in each cell line. No significant difference was detected between patient-related mutations and WT. Only for the m11 mutant protein the association to stress granules was significantly reduced (p=0.0064), indicating the requirement of RNA binding for stress-granule association. P-value were calculated using unpaired two-sided students t.test of mutant compared to WT Ctrl.

Processing-body association of hsPURA in HeLa cells is impaired by patient-derived mutations.

A P-bodies were identified by immunostaining against DCP1A and against the N-terminal FLAG-tag of overexpressed hsPURA FL. Whereas their individual staining is shown in white, the overlay of DCP1A and FLAG is shown in magenta and yellow, respectively. Nuclei were stained with DAPI (blue). All cell lines show P-body formation, marked with red arrows in the zoomed area.

B Box-Whiskers plots of quantification of intensities of hsPURA staining in P-bodies above cytoplasmic background levels. For each cell line, three biological replicates (indicated by different icons) with each 100 P-bodies were quantified, and Box-Whiskers-Plot generated for each cell line. Significantly lower P-body localization was observed for mutant K97E (p = 0.0085), F2333del (P = 0.025) and m11 (p =0.011) variants of hsPURA. P-value were calculated using unpaired two-sided students t.test of mutant compared to WT Ctrl.

Crystal structure of C-terminal PUR domain from hsPURA and effects of mutations on its function.

A Crystal structure of hsPURA III at 1.7 Å resolution (PDB ID: 8CHW). Two hsPURA III molecules (magenta and grey, respectively) form a homodimer. Like other PUR domains (e.g. hsPURA I-II), hsPURA repeat III consists of four β-sheets and one α-helix.

B In electrophoretic mobility shift assays (EMSA) the interaction of hsPURA FL and hsPURA III with a 24-mer (CGG)8 RNA fluorescently labeled with Cy5 fluorophore at 5’end was observed. The amount of the RNA was kept constant at 8 nM while the protein concentration increased from 0 to 16 μM. The unbound RNA used for quantification (see E) is indicated with red arrows.

C Apparent affinities derived from EMSAs (C) indicate that the C-terminal PUR domain is also able to interact with the nucleic acids. Pairwise t-tests of the hsPURA fragments showed significantly lower RNA binding compared to the full-length protein (hsPURA III: p = 9.6E-4). Three replicates were measured for each experiment and protein variant, and the standard deviations have been calculated and shown as bars.

D, E Strand-separation activity of hsPURA. The graphs show the averaged values of three independent experiments as dots with standard deviations as error bars.

D The hsPURA III fragment shows sigmoidal increase of the ssDNA concentration measured as a fluorescent signal. For the quantification of the sigmoidal curves, we utilized Boltzmann function implemented in the Origin software. Calculated x0, which corresponds to the Km in this assay yields 4.26 ± 0.74 µM.

E Strand-separation activity of full-length hsPURA was calculated with Michaelis-Menten kinetic, yielding an average Km value of 0.83 ± 0.05 µM, respectively. For both hsPURA samples at least three independent measurements have been performed.

F NanoBRET experiments in HEK293 cells with different hsPURA fragment-expressing constructs. Milli-BRET units (mBU) were measured for dimerization of hsPURA I-III with hsPURA I-III, hsPURA I-III F233del, and hsPURA I-III R245P. Pairwise t-tests of the mutant hsPURA I-III versions F233del and R245P yielded significantly lower signals compared to the wild-type protein (hsPURA I-III F233del: p = 3.3E-4; hsPURA I-III R245P: p = 1.5E-7), indicating impaired interactions between the proteins. Black horizontal line shows reference of mBU obtained for hsPURA I-II as negative control. Of note, since the BRET signal is the ratio of donor and acceptor signal, it does not require normalization for expression levels. Asterisks in (B) and (G) indicate significance level: *** for p ≤ 0.001.

Structure and nucleic-acids interaction of the N-terminal PUR domain.

A Crystal structure of wild-type hsPURA repeat I (green) and II (blue) at 1.95 Å resolution. Protein fragment for which no electron density was visible is shown as a grey dotted line.

B Ribbon presentation of the overlay of all four chains (shown in different colors) of hsPURA I-II in the asymmetric unit of the crystal. There are three regions showing greater differences in folding between these individual molecules, indicating that they are flexible and likely adopt several conformations also in solution.

C In electrophoretic mobility shift assays, (EMSA) the interaction of different hsPURA I-II variants with 24-mer RNA (CGG)8 labeled with Cy5-fluorophore was observed. The amount of labeled RNA was kept constant at 8 nM while the protein concentration increased from 0 to 16 μM. The unbound RNA was used for quantification (see D) and is indicated with the red arrow. Above the unbound RNA a second band of free RNA is visible, which might constitute a different conformation or RNA dimer.

D Quantification of the RNA interactions of different hsPURA I-II variants from EMSAs shown in (C). The RNA-binding affinity of wild-type hsPURA was normalized to 100%. Pairwise t-test was used to assess differences in RNA binding compared to the hsPURA I-II. The mutants K97E and m11 showed significantly lower relative affinities (p = 1.5E-05 and p = 4.6E-09, respectively). In contrast, the mutation R140P did not alter binding affinity (p = 0.5).

E Strand-separation activity of different hsPURA variants. For each hsPURA sample at least three measurements have been performed. The graphs show the averaged values as points as well as the standard deviations as error bars. The strand-separation activity shows linear increase within the used concentration range.

F Quantitative representation of strand-separating activity of the hsPURA I-II variants. The strand-separating activity of hsPURA I-II was quantified from the slope in (E) and normalized to 100%. Except for R140P (p = 0.07), pairwise t-tests of the hsPURA I-II mutants showed significantly lower relative activity (K97E p = 3.9E-16 and m11 p = 9.2E-16) than hsPURA I-II. For each experiment and each protein variant three replicates were measured, the standard deviations were calculated and are shown as bars. Asterisks in (D) and (F) indicate significance level: * for p ≤ 0.05, *** for p ≤ 0.001; n.s. for p > 0.05.

Structural analysis of hsPURA I-II bearing the K97E patient mutation.

A Circular dichroism (CD) spectroscopic analyses of hsPURA I-II, K97E, and R140P mutant. Shown are the mean of three CD measurements for each of the proteins (n = 3). The spectra of hsPURA I-II K97E show a very different profile, indicating an altered fold of this mutant compared to the wild-type form.

B, C Crystal structures of hsPURA K97E repeat I (green) and II (blue) at 2.45 Å resolution, showing a high overall similarity to hsPURA (Fig 4A). Two independent hsPURA I-II K97E chains (A and B) in the asymmetric unit are shown in (B) and (C), respectively. The mutated amino acid K97E is indicated as red sticks.

D, E Positional shifts of amino acids in β5 (D) and β6 (E) strands of the chain B in the crystal structure of hsPURA I-II K97E. 2Fo-Fc electron density map (contour 1σ) is shown for the selected fragment of β5 and β6 strands in chain B (light pink). The superimposed chain A (grey) shows a register shift in chain B by +1 amino acid in the β5 strand and by +2 in the β6 strand.

F NMR experiment with wild-type hsPURA I-II and hsPURA I-II K97E. Overlay of the 1H, 15N-HSQC spectra of hsPURA I-II (red) and K97E (black). Blue boxes indicate examples of changes between both spectra.