Fig. 4: SR-phosphorylation modulates RNA-induced condensation of the nucleocapsid protein.

a MD simulations of SR/polyU-interaction. The number of contacts per peptide with a 20 base length polyU is shown in the top graph for the non-phosphorylated and fully phosphorylated SR-peptide as the average and standard deviation of five independent simulations. Snapshots of both peptides are shown. b ¹H 1D experiments of the SR-peptide (1766.8 Da), SPRK1 single-phosphorylated peptide (SR(1 P); 1846.9 Da according to mass spectrometry) and SPRK1 double-phosphorylated peptide (SR(2 P); 1926.9 Da according to mass spectrometry; Supplementary Fig. 8) at three different concentrations of polyU (0, 300 and 1500 nM). The spectral regions, in which the signals of polyU, the backbone H^Ns of unmodified residues and phosphorylated serines are located, are marked. The positive charges of the SR-peptide are compensated by the polyU negative charges at around 300 nM. c Turbidity at 350 nm of solutions of non-phosphorylated (blue) and SRPK1-phosphorylated (red) N^SARS-CoV-2 in 20 mM NaPi, pH 7.5, at 30 µM protein concentration and increasing concentrations of polyU. Average values from three independent measurements are shown. Error bars, std. An SDS-Page gel (insert) displays a band shift due to SRPK1-phosphorylation. d Decreased RNA recruitment into polyU-induced droplets of N^SARS-CoV-2 upon phosphorylation with SRPK1 kinase. Mean values and standard deviation are displayed (n = 100 droplets). Two-sided t test with P value set to < 0.05 for statistical significance, *** < 0.001, ** < 0.002 and * < 0.033, ns < 0.12. e FRAP of N^SARS-CoV-2 (blue) and SRPK1-phosphorylated N^SARS-CoV-2 (red) inside of polyU-induced droplets. Error bars represent the standard deviation for averaged 8 and 10 curves for unmodified and phosphorylated N^SARS-CoV-2, respectively. Representative micrographs of N^SARS-CoV-2 (top) and SRPK1-phosphorylated N^SARS-CoV-2 droplets (bottom) before bleaching, after bleaching and at the end of recovery are displayed to the right. Partition coefficients of 12.2 ± 2.8 and 4.3 ± 1.2 were calculated for N^SARS-CoV-2 and phosphorylated N^SARS-CoV-2, respectively. Scale bars, 10 µm. f Association of unmodified (N^SARS-CoV-2, left panels) and phosphorylated (phosphoN^SARS-CoV-2, right panels) nucleocapsid protein of SARS-CoV-2 with stress granules in HeLa cells, micrographs are representative of two independent biological replicates. Scale bar 20 µm, 5 µm in inset.