Peptides that Mimic RS repeats modulate phase separation of SRSF1, revealing a reliance on combined stacking and electrostatic interactions
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eLife assessment
This study convincingly demonstrates that the splicing factor SRSF1 can be solubilized in the presence of short RS or ER-containing peptides, and uses this discovery to determine the solution NMR structure of SRSF1, as well as to map its interactions with RS peptides. These findings are important in that SR proteins are key regulators of alternative splicing but their study has been greatly hampered by their low solubility. The development of a general method that allows the structural and biochemical analysis of SR proteins in solution will have broad applications.
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Abstract
Phase separation plays crucial roles in both sustaining cellular function and perpetuating disease states. Despite extensive studies, our understanding of this process is hindered by low solubility of phase-separating proteins. One example of this is found in SR and SR-related proteins. These proteins are characterized by domains rich in arginine and serine (RS domains), which are essential to alternative splicing and in vivo phase separation. However, they are also responsible for a low solubility that has made these proteins difficult to study for decades. Here, we solubilize the founding member of the SR family, SRSF1, by introducing a peptide mimicking RS repeats as a co-solute. We find that this RS-mimic peptide forms interactions similar to those of the protein’s RS domain. Both interact with a combination of surface-exposed aromatic residues and acidic residues on SRSF1’s RNA Recognition Motifs (RRMs) through electrostatic and cation-pi interactions. Analysis of RRM domains from human SR proteins indicates that these sites are conserved across the protein family. In addition to opening an avenue to previously unavailable proteins, our work provides insight into how SR proteins phase separate and participate in nuclear speckles.
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eLife assessment
This study convincingly demonstrates that the splicing factor SRSF1 can be solubilized in the presence of short RS or ER-containing peptides, and uses this discovery to determine the solution NMR structure of SRSF1, as well as to map its interactions with RS peptides. These findings are important in that SR proteins are key regulators of alternative splicing but their study has been greatly hampered by their low solubility. The development of a general method that allows the structural and biochemical analysis of SR proteins in solution will have broad applications.
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Reviewer #1 (Public Review):
The manuscript seeks to address a major limitation in the study of SFRS1, a critical and well-studied alternative splicing factor. Specifically, this protein is insoluble at high concentrations due to liquid phase separation driven by the RS domain. This work tests the hypothesis that short RS repeat peptides might compete with intermolecular interactions that drive phase separation, thus solubilizing the protein sufficiently for biochemical and structural studies. The data convincingly show that short peptides with RS, ER, or DR repeats can render recombinant SRSF1 soluble. Moreover, the authors present well-resolved and assignable NMR spectra of SRSF1 dissolved with RS8 as a co-solute. Finally, the authors use paramagnetic relaxation enhancement experiments to map interactions between RS8 and SFRS1, which …
Reviewer #1 (Public Review):
The manuscript seeks to address a major limitation in the study of SFRS1, a critical and well-studied alternative splicing factor. Specifically, this protein is insoluble at high concentrations due to liquid phase separation driven by the RS domain. This work tests the hypothesis that short RS repeat peptides might compete with intermolecular interactions that drive phase separation, thus solubilizing the protein sufficiently for biochemical and structural studies. The data convincingly show that short peptides with RS, ER, or DR repeats can render recombinant SRSF1 soluble. Moreover, the authors present well-resolved and assignable NMR spectra of SRSF1 dissolved with RS8 as a co-solute. Finally, the authors use paramagnetic relaxation enhancement experiments to map interactions between RS8 and SFRS1, which suggests that the interactions are driven by a combination of ionic interactions between arginine and acidic side chains, and pi-stacking interactions with surface-exposed hydrophobic residues.
The second aspect of this study seeks to identify features of proteins that make them more likely to undergo phase separations. Specifically, the authors use a bioinformatics approach to correlate the presence of RS repeats with the identity of proteins in three available databases of phase-separated material. In addition, the authors use molecular modeling and software tools to predict additional RRM domains that might prone to phase separation by looking specifically for those that are enriched in acidic amino acids with neighboring hydrophobics. These analyses are less convincing -the fold enrichment is small, the sample size decreases by a large amount with increasing repeat length, and it is not clear that the statistical tests performed correctly for multiple hypothesis testing. Moreover, the predictions of the model derived from the computational analyses have not been explicitly tested.
All told, the NMR and PRE data are convincing and the use of short RS, ER, and DR peptides as a co-solute for insoluble SR-domain proteins is novel and clever, but the computational analyses are incomplete and potentially over-interpreted.
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Reviewer #2 (Public Review):
This manuscript describes several general findings that are relevant to multiple fields. First, using bioinformatic sequence analysis the authors show that RNA Recognition Motif (RRM)-containing proteins often contain a domain with a specific amino acid repeat sequence (Arg-Ser, or RS) that is enriched in proteins that form condensates. With this understanding, the authors sought to use high concentrations of Arg in buffers in an attempt to solubilize an RRM-containing protein that has a C-terminal "RS" repeat region (SRSF1) for NMR structural studies. The high salt content of these samples is not ideal for NMR studies; however, the authors found that small peptides that mimic the RS sequence within SRSF1 can enhance the solubility of SRSF1 with more favorable conditions for NMR. Using paramagnetic …
Reviewer #2 (Public Review):
This manuscript describes several general findings that are relevant to multiple fields. First, using bioinformatic sequence analysis the authors show that RNA Recognition Motif (RRM)-containing proteins often contain a domain with a specific amino acid repeat sequence (Arg-Ser, or RS) that is enriched in proteins that form condensates. With this understanding, the authors sought to use high concentrations of Arg in buffers in an attempt to solubilize an RRM-containing protein that has a C-terminal "RS" repeat region (SRSF1) for NMR structural studies. The high salt content of these samples is not ideal for NMR studies; however, the authors found that small peptides that mimic the RS sequence within SRSF1 can enhance the solubility of SRSF1 with more favorable conditions for NMR. Using paramagnetic relaxation enhancement (PRE) NMR, the authors show that an 8 amino acid RS peptide (RS8) interacts with one of the RRM domains in SRSF1, and the addition of RS8 does not abolish inter- and intra-molecular SRSF1 interactions. PRE NMR-based structure calculations provide a visual assessment of the potential interactions between RS8 and SRSF1. Finally, the authors performed bioinformatic and structural analysis of RRM domains, which was facilitated by AlphaFold-calculated structures, and found that surface-exposed aromatic sequence features appear to be conserved among phase-separating RRM domains.
Strengths of the work include the rigorous approach and the impact that solubilizing repeat peptides could have across many different biological fields where structural data on phase-separating proteins is difficult to obtain. The finding that the RS8 peptide can increase SRSF1 solubility and enable high-resolution NMR analysis should inspire other NMR experimentalists to seek out similar peptide-stabilizing co-solutes for their systems. The bioinformatic analysis that indicates surface-exposed aromatic residues are enriched in RRM domains involved in phase separation provides reasonable hypotheses, which are not directly tested using experimental approaches here but lay the foundation for studies that could be tested in future work and should be generally informative to other researchers interested in RNA-binding proteins.
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