Journal: Nucleic Acids Research

Article Title: Molecular mechanisms of biomolecular condensate formation in Drosophila melanogaster siRNA biogenesis

doi: 10.1093/nar/gkaf664

Figure Lengend Snippet: Ago2 IDR forms droplets upon the addition of nucleic acids. ( A ) RNA-induced droplets of Ago2 IDR (50 μM, PS-I buffer) in the presence of fluorescein-labeled bantam dsRNA (scale bar 10 μm). Liquid-like properties of Ago2 IDR /21 bp hairpin dsRNA condensates are confirmed by time series of FRAP experiments (bottom, 50 μM protein, 25 μM RNA, PS-I buffer, bleach spot radius: 3.2 μm, droplet radius: 6.1 μm, scale bar 10 μm). The sequences of the bantam dsRNA and 21 bp hairpin dsRNA are shown in . ( B ) Particle size changes upon the addition of 21 bp hairpin dsRNA to Ago2 IDR observed by DLS. Top: Particle sizes of Ago2 IDR (50 μM, PS-I buffer) before (black) and after adding 21 bp hairpin dsRNA (25 μM, red). Bottom: Particle sizes after addition of 200 mM ammonium acetate to the Ago2 IDR /dsRNA sample. ( C ) Distinct diffusion rates of Ago2 IDR (100 μM, PS-I buffer) without (dilute phase, left) and with 21 bp hairpin dsRNA (100 μM, biphasic, right) by 1D NMR diffusion experiments ( D = 0.4 s, d = 0.004 s, 800 MHz 1 H Larmor frequency), corresponding to the size changes. ( D ) NMR spectral changes of Ago2 IDR upon phase separation. Superimposed tryptophan indole e1 peak in the 1 H– 15 N HSQCs of Ago2 IDR (100 μM, PS-I buffer) without (black, dilute phase), with 21 bp hairpin dsRNA (100 μM, biphasic, red) and after addition of 100 mM NaCl (light blue, top) or the supernatant after spinning out the droplets by centrifugation (blue, bottom). Full spectra are shown in and J. ( E ) Schematic representation of different sub-constructs of Ago2 IDR (asterisk denotes the region with sequence polymorphism, details in ) and their phase separation propensity based on turbidity assays (Fig. ). ( F ) Phase separation propensity of different sub-constructs of Ago2 IDR in the presence of 21 bp hairpin dsRNA (25 μM) by a turbidity assay (OD 600 ) with increasing protein concentrations in PS-I buffer. Error bars represent the standard deviation.

Article Snippet: 2D 1 H, 1 H NOESY experiments (Bruker library: noesygpph19, 900 MHz 1 H Larmor frequency) for the non-labeled dilute phase (Ago2 4-repeat and heparin, respectively) and dense phase (Ago2 4-repeat with heparin) were recorded at NOE mixing times of 150 ms. 3D 15 N-edited NOESY and 13 C-edited NOESY-HMQC experiments of the dilute phase of Ago2 4-repeat (1 mM, PS-I buffer) were recorded at 950 MHz at NOE mixing times of 150 ms. Intermolecular contacts in the dense phase (Ago2 4-repeat with heparin) were identified via 3D ω 1 – 13 C, 15 N-filtered ω 3 – 15 N-edited and ω 1 – 13 C, 15 N-filtered, ω 3 – 13 C-edited NOESY-HSQC experiments, respectively, performed at 28.2 T (1.2 GHz 1 H Larmor frequency) NOE mixing times of 150 ms. To assess the effect of spin diffusion ω 1 – 13 C, 15 N-filtered ω 3 – 13 C-edited NOESY-HSQC were additionally measured at NOE mixing times of 80 and 30 ms, and 2D 1 H, 1 H NOESY experiments were measured at mixing times of 10, 30, 50, 80, 120, 150, 200, 300, 400, and 500 ms (1.2 GHz 1 H Larmor frequency).

Techniques: Labeling, Diffusion-based Assay, Centrifugation, Construct, Sequencing, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Molecular mechanisms of biomolecular condensate formation in Drosophila melanogaster siRNA biogenesis

doi: 10.1093/nar/gkaf664

Figure Lengend Snippet: The dense phase of Ago2 4-repeat shows characteristic NMR chemical shifts and significantly increased rigidity. ( A ) Particle size changes upon the addition of 21 bp hairpin dsRNA or heparin to Ago2 4-repeat are observed by DLS. Left: Ago2 4-repeat (500 μM, PS-I buffer) without (black) and with 21 bp hairpin dsRNA (25 μM, red). Right: Ago2 4-repeat (100 μM, PS-I buffer) without (black) and with heparin (0.2 mg/ml, red). ( B ) The residual effect of phase separation was detected by NMR. Comparison of the 1 H– 15 N HSQC spectra of Ago2 4-repeat (100 μM, dilute phase, black), Ago2 4-repeat (100 μM) with 0.2 mg/ml heparin to induce phase separation (biphasic, orange), a condensed phase sample of Ago2 4-repeat with heparin (∼20 mM, dense phase, red), the supernatant after centrifugation of the biphasic sample (centrifuged, blue), and after addition of 2 mg/ml heparin (high heparin concentration, light blue, PS-I buffer). ( C ) Diffusion rates of Ago2 4-repeat in the dilute phase (420 μM), biphase (420 μM, 1 mg/ml heparin), and dense phase (∼ 20 mM, PS-I buffer) by 2D 1 H- 13 C NMR diffusion experiments (900 MHz 1 H Larmor frequency). Left: Overlay of the 1 H- 13 C NMR spectra at increasing gradient strength for residues E11 H-C γ and R7/R17 H-C δ for the corresponding phases. Right: Normalized intensity ratio as a function of gradient strength for E11 and R7/R17 in the corresponding phases (dilute phase: black, biphase dilute: blue, biphase dense: orange, dense phase: red). ( D ) Comparison of the backbone dynamics of Ago2 4-repeat in the dilute (black, 850 μM, PS-I buffer) or dense (with heparin, red) phases by 15 N NMR relaxation experiments ({ 1 H}- 15 N heteronuclear nuclear Overhauser effect (hetNOE), T 2 (900 MHz 1 H Larmor frequency), and T 1ρ (950 MHz 1 H Larmor frequency). The error margins for the hetNOE experiment are obtained from spectral noise and the errors for T 2 and T 1ρ are obtained from fitting to the exponential function.

Article Snippet: 2D 1 H, 1 H NOESY experiments (Bruker library: noesygpph19, 900 MHz 1 H Larmor frequency) for the non-labeled dilute phase (Ago2 4-repeat and heparin, respectively) and dense phase (Ago2 4-repeat with heparin) were recorded at NOE mixing times of 150 ms. 3D 15 N-edited NOESY and 13 C-edited NOESY-HMQC experiments of the dilute phase of Ago2 4-repeat (1 mM, PS-I buffer) were recorded at 950 MHz at NOE mixing times of 150 ms. Intermolecular contacts in the dense phase (Ago2 4-repeat with heparin) were identified via 3D ω 1 – 13 C, 15 N-filtered ω 3 – 15 N-edited and ω 1 – 13 C, 15 N-filtered, ω 3 – 13 C-edited NOESY-HSQC experiments, respectively, performed at 28.2 T (1.2 GHz 1 H Larmor frequency) NOE mixing times of 150 ms. To assess the effect of spin diffusion ω 1 – 13 C, 15 N-filtered ω 3 – 13 C-edited NOESY-HSQC were additionally measured at NOE mixing times of 80 and 30 ms, and 2D 1 H, 1 H NOESY experiments were measured at mixing times of 10, 30, 50, 80, 120, 150, 200, 300, 400, and 500 ms (1.2 GHz 1 H Larmor frequency).

Techniques: Comparison, Centrifugation, Concentration Assay, Diffusion-based Assay

Journal: Nucleic Acids Research

Article Title: Molecular mechanisms of biomolecular condensate formation in Drosophila melanogaster siRNA biogenesis

doi: 10.1093/nar/gkaf664

Figure Lengend Snippet: Diverse protein interactions and arginine-mediated electrostatic interactions play a role in the dense phase of Ago2 4-repeat and heparin. ( A ) Schematic representation of the intermolecular protein contacts visible in a filtered NOESY experiment. ( B ) Intermolecular contacts in the Ago2 4-repeat dense phase. Left: Strips of selected residues of the ω 1 – 13 C, 15 N-filtered ω 3 – 15 N-edited NOESY, and ω 1 – 13 C, 15 N-filtered ω 3 – 13 C-edited NOESY-HSQC (150 ms NOE mixing time, 1.2 GHz 1 H Larmor frequency) of Ago2 4-repeat dense phase (gray). Intermolecular NOE cross peaks for which assignment was possible are labeled in red. Right: Type of interactions suggested by the intermolecular contacts to be present in the condensates. ( C ) Intensities of intermolecular NOEs ( ω 1 – 13 C, 15 N-filtered ω 3 – 13 C-edited NOESY-HSQC, mixing time 80 ms) of Arginine H δ and Tyrosine H δ to 1 H, 13 C strips show a wide range of contacts in the condensed phase. Stacked bars represent different residue types. NOE intensities are corrected for the number of residues in amino acid sequence. Only unambiguously assigned residues are reported. ( D ) Effect of amino acid composition on Ago2 4-repeat phase separation. Top: Sequence of Ago2 4-repeat . All glutamates or serines or histidines or glutamines or tyrosines or arginines in the protein sequence are mutated to glycine, all histidines are mutated to tyrosines or lysines, all glutamines or arginines are mutated to alanine, all arginines are mutated to lysines or the first or second arginines in each repeat (R7 or R17) are mutated to glycines. Bottom: Fluorescent microscopy images of phase separation of Ago2 4-repeat WT and mutants (800 μM, PS-I buffer) with heparin (2 mg/ml) at room temperature (scale bar 10 μm). ( E ) Quantitative analysis of the effect of mutations in Ago2 4-repeat on condensate formation. Sedimentation assay of Ago2 4-repeat WT and mutants (800 μM, PS-I buffer) with heparin (2 mg/ml) at 25°C (black) and 5°C (gray). Error bars represent the standard deviation of three replicates from distinct samples.

Article Snippet: 2D 1 H, 1 H NOESY experiments (Bruker library: noesygpph19, 900 MHz 1 H Larmor frequency) for the non-labeled dilute phase (Ago2 4-repeat and heparin, respectively) and dense phase (Ago2 4-repeat with heparin) were recorded at NOE mixing times of 150 ms. 3D 15 N-edited NOESY and 13 C-edited NOESY-HMQC experiments of the dilute phase of Ago2 4-repeat (1 mM, PS-I buffer) were recorded at 950 MHz at NOE mixing times of 150 ms. Intermolecular contacts in the dense phase (Ago2 4-repeat with heparin) were identified via 3D ω 1 – 13 C, 15 N-filtered ω 3 – 15 N-edited and ω 1 – 13 C, 15 N-filtered, ω 3 – 13 C-edited NOESY-HSQC experiments, respectively, performed at 28.2 T (1.2 GHz 1 H Larmor frequency) NOE mixing times of 150 ms. To assess the effect of spin diffusion ω 1 – 13 C, 15 N-filtered ω 3 – 13 C-edited NOESY-HSQC were additionally measured at NOE mixing times of 80 and 30 ms, and 2D 1 H, 1 H NOESY experiments were measured at mixing times of 10, 30, 50, 80, 120, 150, 200, 300, 400, and 500 ms (1.2 GHz 1 H Larmor frequency).

Techniques: Labeling, Residue, Sequencing, Microscopy, Sedimentation, Standard Deviation