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Lysine/RNA-interactions drive and regulate biomolecular condensation

Ukmar-Godec, Tina, Hutten, Saskia, Grieshop, Matthew P., Rezaei-Ghaleh, Nasrollah, Cima-Omori, Maria-Sol, Biernat, Jacek, Mandelkow, Eckhard, Söding, Johannes, Dormann, Dorothee, Zweckstetter, Markus·2019·Nature Communications
DOI 10.1038/s41467-019-10792-yPMC PMC6606616
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Abstract

Cells form and use biomolecular condensates to execute biochemical reactions. The molecular properties of non-membrane-bound condensates are directly connected to the amino acid content of disordered protein regions. Lysine plays an important role in cellular function, but little is known about its role in biomolecular condensation. Here we show that protein disorder is abundant in protein/RNA granules and lysine is enriched in disordered regions of proteins in P-bodies compared to the entire human disordered proteome. Lysine-rich polypeptides phase separate into lysine/RNA-coacervates that are more dynamic and differ at the molecular level from arginine/RNA-coacervates. Consistent with the ability of lysine to drive phase separation, lysine-rich variants of the Alzheimer’s disease-linked protein tau undergo coacervation with RNA in vitro and bind to stress granules in cells. Acetylation of lysine reverses liquid–liquid phase separation and reduces colocalization of tau with stress granules. Our study establishes lysine as an important regulator of cellular condensation.

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Figures

Fig. 1 Disordered protein regions in P-bodies are rich in lysine. a Distribution of the fraction of disorder predicted by IUpred for P-body proteins (top) and the entire human proteome (bottom). b Box plot of log2-fold enrichment of amino acids in disordered regions of P-bodies relative to disordered regions of the entire human proteome. Boxes and whiskers show, from bottom to top, 5%, 25%, 50%, 75%, and 95% quantiles. Red/blue: positively/negatively charged residues. p-values < 10−5:*, < 10–10:**, < 10–20:***. c Log2 enrichment (red) or depletion (blue) of dipeptides in disordered regions of P-bodies relative to disordered regions of the entire human proteome. First residue on x-axis, second on y-axis. Size of circles: total probability of dipeptide (sum = 1). d Same charges cluster together in P-bodies, while opposite charges do not. Top: Total counts of amino acid K at position x relative to an E at position 0 (first residue) in disordered regions of P-bodies. Virtually no clustering is observed. Middle and bottom: Same for K/K and E/E instead of K/E. Residues of same charge are enriched within + /− 5 residues of each other. Source data are provided as a Source Data file
Fig. 1 Disordered protein regions in P-bodies are rich in lysine. a Distribution of the fraction of disorder predicted by IUpred for P-body proteins (top) and the entire human proteome (bottom). b Box plot of log2-fold enrichment of amino acids in disordered regions of P-bodies relative to disordered regions of the entire human proteome. Boxes and whiskers show, from bottom to top, 5%, 25%, 50%, 75%, and 95% quantiles. Red/blue: positively/negatively charged residues. p-values < 10−5:*, < 10–10:**, < 10–20:***. c Log2 enrichment (red) or depletion (blue) of dipeptides in disordered regions of P-bodies relative to disordered regions of the entire human proteome. First residue on x-axis, second on y-axis. Size of circles: total probability of dipeptide (sum = 1). d Same charges cluster together in P-bodies, while opposite charges do not. Top: Total counts of amino acid K at position x relative to an E at position 0 (first residue) in disordered regions of P-bodies. Virtually no clustering is observed. Middle and bottom: Same for K/K and E/E instead of K/E. Residues of same charge are enriched within + /− 5 residues of each other. Source data are provided as a Source Data fileCC BY · PubMed Central
Fig. 2 Complex coacervation of lysine-rich polypeptides. a Amino acid sequences of the lysine-rich (K3) and arginine-rich peptide (R3) along with a schematic of complex coacervation upon addition of RNA. b (left) Comparison of the turbid (phase-separated) peptide-containing sample and the control sample/buffer. (Right) DIC image of a pair of K3/RNA liquid droplets undergoing fusion. The concentration of polyU RNA was 0.5 mg ml−1 and K3 was 1 mM. c Turbidity of K3 with tRNA (0.5 mg ml−1) and R3 with tRNA (0.5 mg ml−1) samples at different pH values and peptide concentrations (0.1 mM, 0.5 mM, and 1 mM). Reported turbidity values represent the average of a triplicate of measurements for each sample. d Distribution of droplet diameters after 5 and 20 min with the corresponding fluorescence images. The concentration of polyU RNA was 1 mg ml−1. e DIC and fluorescence images depicting the tRNA concentration-dependence of K3 (top) and R3 (bottom) coacervation. Scale bars, 10 μm. f Normalized FRAP recovery curves (left) for K3/RNA-coacervates, K2R1/RNA-coacervates and R3/RNA-coacervates with the corresponding FRAP images (right; recovery image 27.2 s after bleaching; scale bar 1 μm). Fits to the experimental data assuming an exponential recovery (K3, K2R1; solid line) and diffusion-controlled recovery (R3; Axelrod model, dash-dotted line). The concentration of K3, K2R1, R3, and polyU RNA in DIC, fluorescence imaging, and FRAP were identical and equal to 1 mM K3/K2R1/R3 and 1 mg ml−1 polyU RNA, in 50 mM HEPES, pH 7.4. TMR-K3, TMR-K2R1 and TMR-R3 were used as specified in the methods section. Error bars in c and f correspond to ± SD. Source data are provided as a Source Data file
Fig. 2 Complex coacervation of lysine-rich polypeptides. a Amino acid sequences of the lysine-rich (K3) and arginine-rich peptide (R3) along with a schematic of complex coacervation upon addition of RNA. b (left) Comparison of the turbid (phase-separated) peptide-containing sample and the control sample/buffer. (Right) DIC image of a pair of K3/RNA liquid droplets undergoing fusion. The concentration of polyU RNA was 0.5 mg ml−1 and K3 was 1 mM. c Turbidity of K3 with tRNA (0.5 mg ml−1) and R3 with tRNA (0.5 mg ml−1) samples at different pH values and peptide concentrations (0.1 mM, 0.5 mM, and 1 mM). Reported turbidity values represent the average of a triplicate of measurements for each sample. d Distribution of droplet diameters after 5 and 20 min with the corresponding fluorescence images. The concentration of polyU RNA was 1 mg ml−1. e DIC and fluorescence images depicting the tRNA concentration-dependence of K3 (top) and R3 (bottom) coacervation. Scale bars, 10 μm. f Normalized FRAP recovery curves (left) for K3/RNA-coacervates, K2R1/RNA-coacervates and R3/RNA-coacervates with the corresponding FRAP images (right; recovery image 27.2 s after bleaching; scale bar 1 μm). Fits to the experimental data assuming an exponential recovery (K3, K2R1; solid line) and diffusion-controlled recovery (R3; Axelrod model, dash-dotted line). The concentration of K3, K2R1, R3, and polyU RNA in DIC, fluorescence imaging, and FRAP were identical and equal to 1 mM K3/K2R1/R3 and 1 mg ml−1 polyU RNA, in 50 mM HEPES, pH 7.4. TMR-K3, TMR-K2R1 and TMR-R3 were used as specified in the methods section. Error bars in c and f correspond to ± SD. Source data are provided as a Source Data fileCC BY · PubMed Central
Fig. 3 NMR spectroscopy and temporal stability of lysine-driven condensation. a 1D 1H NMR spectra of the lysine-rich peptide K3 (1 mM; blue), K3 with polyU RNA forming liquid droplets (1 mM K3 and 1 mg ml−1 polyU RNA; black) and the same sample after 24 h (red), as well as polyU RNA alone (1 mg ml−1; green). Peptide NMR signals, which are characteristic for the interaction with polyU RNA, are marked with asterisks. b 2D 1H−13C HSQC NMR spectrum of K3 (blue) and K3 with polyU RNA (orange). Cross-peaks were labeled according to amino acid-specific chemical shift values. The two K3/RNA-specific NMR signals at 1H frequencies of 0.53 and 0.59 ppm (marked with asterisks in a) were also present in the 2D 1H-13C HSQC NMR spectrum and have identical 13C chemical shifts as the two leucine Cδ resonances of K3 in the absence of polyU RNA. c Temporal stability of K3 droplets as monitored by the time-dependent decrease of NMR signals. (Top) 1D 1H NMR spectra of K3 at 0, 1, 2, 3, 4, 5, 6, and 7 h with K3/RNA-specific signals labeled from a–e. (Bottom) Time-dependent decrease in normalized signal intensity of K3/RNA-specific signals. Error bars are based on signal-to-noise ratios in the NMR spectra. The peptide solution became clear after ~ 7 h. d DIC micrographs of a sample containing K3 (1 mM) and polyU RNA (1 mg ml−1) over a period of 8 h. Liquid droplets disappeared after about 7–8 h. Scale bar, 10 μm
Fig. 3 NMR spectroscopy and temporal stability of lysine-driven condensation. a 1D 1H NMR spectra of the lysine-rich peptide K3 (1 mM; blue), K3 with polyU RNA forming liquid droplets (1 mM K3 and 1 mg ml−1 polyU RNA; black) and the same sample after 24 h (red), as well as polyU RNA alone (1 mg ml−1; green). Peptide NMR signals, which are characteristic for the interaction with polyU RNA, are marked with asterisks. b 2D 1H−13C HSQC NMR spectrum of K3 (blue) and K3 with polyU RNA (orange). Cross-peaks were labeled according to amino acid-specific chemical shift values. The two K3/RNA-specific NMR signals at 1H frequencies of 0.53 and 0.59 ppm (marked with asterisks in a) were also present in the 2D 1H-13C HSQC NMR spectrum and have identical 13C chemical shifts as the two leucine Cδ resonances of K3 in the absence of polyU RNA. c Temporal stability of K3 droplets as monitored by the time-dependent decrease of NMR signals. (Top) 1D 1H NMR spectra of K3 at 0, 1, 2, 3, 4, 5, 6, and 7 h with K3/RNA-specific signals labeled from a–e. (Bottom) Time-dependent decrease in normalized signal intensity of K3/RNA-specific signals. Error bars are based on signal-to-noise ratios in the NMR spectra. The peptide solution became clear after ~ 7 h. d DIC micrographs of a sample containing K3 (1 mM) and polyU RNA (1 mg ml−1) over a period of 8 h. Liquid droplets disappeared after about 7–8 h. Scale bar, 10 μmCC BY · PubMed Central
Fig. 4 Influence of tau domain structure and co-factors on LLPS. a Domain organization of hTau40, K25, and K18. N1 and N2 are the two inserts not present in K25, P1, and P2 the two proline-rich regions, and R1–R4 the four pseudo-repeats. The pI and the number of lysine (K) and arginine (R) residues in hTau40, K25 and K18 are indicated. b DIC and fluorescence micrographs of hTau40, K25, and K18 solutions in the presence of different co-factors/buffer conditions. The concentration of the proteins was 50 μM. c Droplet size distribution of K18 (top) and hTau40 (bottom) after five and 20 min alongside corresponding fluorescence images. K18 (50 μM) with 125 µg ml−1 polyU RNA in 25 mM HEPES, pH 7.4, was used. For hTau40 (50 μM), the same buffer was used with 10% dextran (and no RNA). Scale bar, 10 μm
Fig. 4 Influence of tau domain structure and co-factors on LLPS. a Domain organization of hTau40, K25, and K18. N1 and N2 are the two inserts not present in K25, P1, and P2 the two proline-rich regions, and R1–R4 the four pseudo-repeats. The pI and the number of lysine (K) and arginine (R) residues in hTau40, K25 and K18 are indicated. b DIC and fluorescence micrographs of hTau40, K25, and K18 solutions in the presence of different co-factors/buffer conditions. The concentration of the proteins was 50 μM. c Droplet size distribution of K18 (top) and hTau40 (bottom) after five and 20 min alongside corresponding fluorescence images. K18 (50 μM) with 125 µg ml−1 polyU RNA in 25 mM HEPES, pH 7.4, was used. For hTau40 (50 μM), the same buffer was used with 10% dextran (and no RNA). Scale bar, 10 μmCC BY · PubMed Central
Fig. 5 Dynamics of tau in tau/RNA droplets. a FRAP images (recovery image 129 s after bleaching; scale bar 1 μm). b Normalized FRAP recovery curves for hTau40 (50 µM; gray) and K18 (50 µM; red) droplets in the presence of 40 µg ml−1 polyU RNA (25 mM HEPES, pH 7.4). Symbols with error bars depict experimental results; black lines are fits assuming diffusion-controlled recovery (i.e., the Axelrod model). Error bars in b correspond to ± SD. Source data are provided as a Source Data file
Fig. 5 Dynamics of tau in tau/RNA droplets. a FRAP images (recovery image 129 s after bleaching; scale bar 1 μm). b Normalized FRAP recovery curves for hTau40 (50 µM; gray) and K18 (50 µM; red) droplets in the presence of 40 µg ml−1 polyU RNA (25 mM HEPES, pH 7.4). Symbols with error bars depict experimental results; black lines are fits assuming diffusion-controlled recovery (i.e., the Axelrod model). Error bars in b correspond to ± SD. Source data are provided as a Source Data fileCC BY · PubMed Central
Fig. 6 Acetylation reverses lysine-driven coacervation. a Cartoon representation illustrating acetylation of lysine-rich polypeptides by lysine acetyltransferase (KAT) in the presence of acetyl-Coenzyme A (AcCoA) (left) and its influence on lysine/RNA coacervation (right). b Mass spectra of K18 and hTau40 samples before (top) and after acetylation by CREB (bottom). Identified masses (in Da) are indicated. c DIC and fluorescence images show dissolution of K18/RNA (top; 50 µM K18, 125 µg ml−1 polyU RNA in 25 mM HEPES, pH 7.4) and hTau40/dextran (bottom; 50 µM hTau40, 10% dextran in 25 mM HEPES, pH 7.4) droplets in the presence of the acetyltransferase CREB and AcCoA. Time-dependent changes in droplet numbers were quantified (left). Experiments were performed three times for each protein sample. d, e Turbidity measurements of K18 (50 µM; d), as well as arginine- (R3; 1 mM) and lysine-rich (K2 and K3; 1 mM) peptides (e), which underwent LLPS through addition of polyU RNA (125 µg ml-1 for K18, 0.5 mg ml-1 for the peptides), in the presence of either CREB/p300 or AcCoA, or both the enzyme and AcCoA. In the middle, photographs of the respective K18 samples after 60 min are depicted. Reported values represent an average of three independent samples. Error bars in d and e correspond to ± SD. Source data are provided as a Source Data file
Fig. 6 Acetylation reverses lysine-driven coacervation. a Cartoon representation illustrating acetylation of lysine-rich polypeptides by lysine acetyltransferase (KAT) in the presence of acetyl-Coenzyme A (AcCoA) (left) and its influence on lysine/RNA coacervation (right). b Mass spectra of K18 and hTau40 samples before (top) and after acetylation by CREB (bottom). Identified masses (in Da) are indicated. c DIC and fluorescence images show dissolution of K18/RNA (top; 50 µM K18, 125 µg ml−1 polyU RNA in 25 mM HEPES, pH 7.4) and hTau40/dextran (bottom; 50 µM hTau40, 10% dextran in 25 mM HEPES, pH 7.4) droplets in the presence of the acetyltransferase CREB and AcCoA. Time-dependent changes in droplet numbers were quantified (left). Experiments were performed three times for each protein sample. d, e Turbidity measurements of K18 (50 µM; d), as well as arginine- (R3; 1 mM) and lysine-rich (K2 and K3; 1 mM) peptides (e), which underwent LLPS through addition of polyU RNA (125 µg ml-1 for K18, 0.5 mg ml-1 for the peptides), in the presence of either CREB/p300 or AcCoA, or both the enzyme and AcCoA. In the middle, photographs of the respective K18 samples after 60 min are depicted. Reported values represent an average of three independent samples. Error bars in d and e correspond to ± SD. Source data are provided as a Source Data fileCC BY · PubMed Central
Fig. 7 Acetylation weakens binding of tau to RNA. a Domain organization of hTau40. The location of its 44 lysine residues is marked by arrows. b Mass spectra of 15N-labeled hTau40 (left) and 15N-labeled hTau40 acetylated by CREB (right). Identified masses (in Da) are indicated. Tau is acetylated at up to 14 sites. c Superposition of selected regions of 2D 1H-15N spectra of hTau40 (top, black) and acetylated hTau40 (bottom, gray), respectively, for increasing molar ratios of tRNA: 0.25 (cyan), 0.5 (orange) and 1 (green). d Averaged 1H,15N chemical shift perturbations (CSP) of resonances in 2D 1H-15N HSQC spectra of hTau40 caused by binding to tRNA (color code as in c). The net charge of different domains of hTau40 is illustrated on top. Arrows mark lysine and arginine residues. e CSP of resonances in 2D 1H-15N HSQC of hTau40 (black) and acetylated hTau40 (gray) caused by the addition of an equimolar concentration of tRNA (see also b)
Fig. 7 Acetylation weakens binding of tau to RNA. a Domain organization of hTau40. The location of its 44 lysine residues is marked by arrows. b Mass spectra of 15N-labeled hTau40 (left) and 15N-labeled hTau40 acetylated by CREB (right). Identified masses (in Da) are indicated. Tau is acetylated at up to 14 sites. c Superposition of selected regions of 2D 1H-15N spectra of hTau40 (top, black) and acetylated hTau40 (bottom, gray), respectively, for increasing molar ratios of tRNA: 0.25 (cyan), 0.5 (orange) and 1 (green). d Averaged 1H,15N chemical shift perturbations (CSP) of resonances in 2D 1H-15N HSQC spectra of hTau40 caused by binding to tRNA (color code as in c). The net charge of different domains of hTau40 is illustrated on top. Arrows mark lysine and arginine residues. e CSP of resonances in 2D 1H-15N HSQC of hTau40 (black) and acetylated hTau40 (gray) caused by the addition of an equimolar concentration of tRNA (see also b)CC BY · PubMed Central
Fig. 8 Influence of tau domains and lysine acetylation on SG association of tau. a Schematic diagram illustrating the semi-permeabilized cell assay. After SG induction by MG132, the plasma membrane of Hela P4 cells is selectively permeabilized using digitonin, cytosolic soluble factors are washed out and subsequently nuclear pore complexes blocked by WGA. Next, Alexa 488-labeled tau proteins are incubated with semi-permeabilized cells to allow binding to SGs and after extensive washing, SGs were subjected to immunostaining for the SG makers G3BP1 and TIA-1. b SG association of K18, K25, and hTau40 (400 nM; degree of labeling (“DOL corrected”) = 3.7), scale bar, 10 μm. In the merge, Alexa 488 fluorescence (green) and TIA-1 staining (red) are shown. c Quantification showing the Log2-transformed mean fluorescence intensity of K18, K25, and hTau40 vs. background (bkgr) in SGs. Four replicates with at least 8–10 cells and ≥ 25 SGs each are shown. Error bars correspond to ± SEM. d SG association of unmodified hTau40 (400 nM; DOL = 1.0) in comparison to acetylated hTau40 (Ac-hTau40) monitored by Alexa 488-fluorescence. In the merge, Alexa 488 fluorescence (green) and TIA-1 staining (red) are shown. e Quantification showing the Log2-transformed mean fluorescence intensity of hTau40 and Ac-hTau40 vs. background in SGs. Four replicates with at least 8–10 cells and ≥ 37 SGs each are shown. Error bars correspond to ± SEM. Please note, that due to the wide range of fluorescence intensities of the recombinant proteins in SGs, different laser settings had to be used in b when compared to d in order to avoid pixel saturation and to ensure ideal representation of the differential SG recruitment of the different proteins. Source data are provided as a Source Data file
Fig. 8 Influence of tau domains and lysine acetylation on SG association of tau. a Schematic diagram illustrating the semi-permeabilized cell assay. After SG induction by MG132, the plasma membrane of Hela P4 cells is selectively permeabilized using digitonin, cytosolic soluble factors are washed out and subsequently nuclear pore complexes blocked by WGA. Next, Alexa 488-labeled tau proteins are incubated with semi-permeabilized cells to allow binding to SGs and after extensive washing, SGs were subjected to immunostaining for the SG makers G3BP1 and TIA-1. b SG association of K18, K25, and hTau40 (400 nM; degree of labeling (“DOL corrected”) = 3.7), scale bar, 10 μm. In the merge, Alexa 488 fluorescence (green) and TIA-1 staining (red) are shown. c Quantification showing the Log2-transformed mean fluorescence intensity of K18, K25, and hTau40 vs. background (bkgr) in SGs. Four replicates with at least 8–10 cells and ≥ 25 SGs each are shown. Error bars correspond to ± SEM. d SG association of unmodified hTau40 (400 nM; DOL = 1.0) in comparison to acetylated hTau40 (Ac-hTau40) monitored by Alexa 488-fluorescence. In the merge, Alexa 488 fluorescence (green) and TIA-1 staining (red) are shown. e Quantification showing the Log2-transformed mean fluorescence intensity of hTau40 and Ac-hTau40 vs. background in SGs. Four replicates with at least 8–10 cells and ≥ 37 SGs each are shown. Error bars correspond to ± SEM. Please note, that due to the wide range of fluorescence intensities of the recombinant proteins in SGs, different laser settings had to be used in b when compared to d in order to avoid pixel saturation and to ensure ideal representation of the differential SG recruitment of the different proteins. Source data are provided as a Source Data fileCC BY · PubMed Central

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Cited by

Metadata

Authors
Ukmar-Godec, T., Hutten, S., Grieshop, M. P., Rezaei-Ghaleh, N., Cima-Omori, M.-S., Biernat, J., Mandelkow, E., Söding, J., Dormann, D., & Zweckstetter, M.
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2019-07-02
Volume
10
Issue
1
ISSN
2041-1723
DOI
10.1038/s41467-019-10792-y
PMC
PMC6606616
Cited by
261
References
13
Language
en
Licence

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

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Bibliographic data: Semantic Scholar, OpenAlex, Crossref.

Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).

Semantic Scholar’s data is described in: Kinney, R., et al. (2023). The Semantic Scholar Open Data Platform. arXiv:2301.10140.