Peptides that Mimic RS repeats modulate phase separation of SRSF1, revealing a reliance on combined stacking and electrostatic interactions
- Department of Chemistry, University of Alabama at Birmingham, United States
Figures
Figure 1 with 1 supplement
A combination of RS repeats and RRM domains is highly correlated with appearance in condensates.
(A) Increased RS repeat length leads to an increased likelihood of appearance in condensates. Percentage of proteins possessing indicated properties that appear in one of three major phase …
Figure 1—figure supplement 1
Increased repeat number and R/S percent composition correlate with increased phase separation.
(A) Amino-acid sequence of the protein SRRM2 with 56 4-mer RS repeats highlighted in yellow. Red serines can be phosphorylated according to the Uniprot database. Deletion of the undeca-repeat (UPR) …
Figure 2 with 1 supplement
SRSF1 phase separation can be reduced using peptides that best mimic its RS repeats in their respective phosphorylation states.
(A) Schematic illustration of solubilizing phase-separating proteins using short peptides. Short peptides compete with RS repetitive regions, disrupting phase separation. (B) Domain architecture of …
Figure 2—figure supplement 1
Repetitive peptides solubilize other proteins containing similar repetitive regions.
(A) Solubilizing effects of 8-mer Arg-Ser (RS8), Glu-Arg (ER8), and Asp-Arg (DR8) were tested on Nob1 (blue bars) and Nop9 (green bars) at peptide concentrations of 50 mM (open bars) and 100 mM …
Figure 3 with 1 supplement
Short peptides are compatible with NMR experiments.
(A) NMR 90 degree pulse width. ER8 is insoluble at 400 mM and therefore its pulse width could not be determined, as indicated by *. (B) 15N-TROSY-HSQC overlay of SRSF1 in 100 mM RS8 and …
Figure 3—figure supplement 1
The buffer 100 mM ER4, 400 mM Arg/Glu, pH 6.4 was effective at solubilizing and optimizing spectral quality for both unphosphorylated and hyperphosphorylated SRSF1 at high enough concentrations for NMR assignment.
(A) Pulse width of various concentrations of ER4 and Arg/Glu. (B) 15N-TROSY-HSQC overlay of 370 µM SRSF1 (blue) and 540 µM phosphorylated SRSF1 (pi-SRSF1, red) in this buffer. (C) The buffer 100 mM …
Figure 4 with 2 supplements
SRSF1 residues involved in interactions with the RS8 peptide are similar to those found in intra-, and homotypic inter-molecular interactions with the RS region.
(A) 15N-TROSY-HSQC overlay of SRSF1 in 50 mM diamagnetic (gray) and 2.5 mM paramagnetic RS8 (green). The intensities of residues close to the probe become diminished. Bleached residues (indicated by …
Figure 4—figure supplement 1
SRSF1 residues involved in interactions with the RS8 peptide are similar to those found in intra-, and homotypic inter-molecular interactions with the RS region.
15N-TROSY-HSQC overlay of samples used in (A) Peptide-SRSF1, (B) Intramolecular, and (C) Intermolecular PRE experiments. Spectra in which the paramagnetic center was active are color coded, and …
Figure 4—figure supplement 2
SRSF1 residues involved in interactions with the RS8 peptide are similar to those found in intra-, and homotypic inter-molecular interactions with the RS region.
PRE of MTSL alone (A), an intramolecular PRE experiment with the MTSL tag on the very C-terminal end of the protein (B), and an intermolecular PRE experiment replicating the conditions of the …
Figure 5 with 2 supplements
Electrostatic and cation-pi interactions are responsible for intermolecular interactions.
Molecular dynamics simulation of SRSF1 with four RS8 peptides.
Figure 5—figure supplement 1
Fitting of MD simulation structure to PRE values.
(A) Sample 10-membered ensemble generated by Xplor-NIH. RRM1 (residues 16–90) is held in place while the N-terminus and linker are allowed full flexibility (shown as transparent cartoons). RRM2 …
Figure 5—figure supplement 2
MD simulation trajectories of SRSF1 ΔRS interacting with four RS8 peptides.
Root mean standard deviation between the simulation starting structure and the structure at a given simulation time (A–E) Interactions between peptides (left) and the SRSF1 ΔRS residues with which …
Figure 6
RRM1 residues responsible for SRSF1 phase separation are conserved throughout the SR protein family.
ClustalX alignment of RRM1 domains of the SR protein family, where yellow indicates identical amino acids, green and blue indicate conserved residues. Black boxes indicate PRE hotspots. Structure of …
Additional files
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Supplementary file 1
Effect of increasing length on phase separation for all possible dipeptide repeat combinations.
Lengths (x) analyzed are x=0, x>2, x>4, x>6, and x>8. p-values were obtained using a two-tailed correlation analysis. A p-value of <0.05 was considered an indicator that increasing repeat length correlated significantly with fraction of proteins found in condensates. A population-based error of was used to identify whether the sample size was large enough to draw conclusions from the data (as discussed in more detail in the methods section). Whether a repeat type passed both p-value and population-based error criteria is indicated in the right-most column.
- https://cdn.elifesciences.org/articles/84412/elife-84412-supp1-v2.xlsx
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Supplementary file 2
Analysis of the effect of increasing repeat length when datasets are size-matched to either n=14 (sheet 1) or n=6 (sheet 2).
Percentage of proteins in condensates was found through Python’s random selection tool 50 different times for each population. Average and standard deviation are indicated at the top of the sheet.
- https://cdn.elifesciences.org/articles/84412/elife-84412-supp2-v2.xlsx
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Supplementary file 3
Contingency tables analyzing the likelihood of RRM domain and RS repat co-occurrence both for proteins in condensates (left) and proteins not in condensates (right).
Values used correspond to those displayed in Figure 1A. p-values were obtained using Fisher’s exact test.
- https://cdn.elifesciences.org/articles/84412/elife-84412-supp3-v2.xlsx
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Supplementary file 4
Contingency tables corresponding to the p-values shown in Figure 1C and Figure 1—figure supplement 1C.
Correlation between the fraction of proteins in condensates and the presence of a threshold number of short repeats or percentage R/S composition. p-values were obtained using Fisher’s exact test.
- https://cdn.elifesciences.org/articles/84412/elife-84412-supp4-v2.xlsx
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Supplementary file 5
Values corresponding to the distance between peptides and hotspot residues at various timepoints in the MD simulation.
Distances (r) correspond to the length between the cysteine to which the paramagnetic center is attached and the NH hydrogen on the backbone of each residue under observation. Distances were measured using the Pymol measurement feature. Residue 42 is near to two peptides. Therefore, two distances are provided for residue 42. Bleached residues are expected to be within 12–15 Å of the paramagnetic center (Iwahara et al., 2007).
- https://cdn.elifesciences.org/articles/84412/elife-84412-supp5-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/84412/elife-84412-mdarchecklist1-v2.docx
