Peptides that Mimic RS repeats modulate phase separation of SRSF1, revealing a reliance on combined stacking and electrostatic interactions

Fargason, Talia; Silva, Naiduwadura Ivon Upekala De; King, Erin; Zhang, Zihan; Paul, Trenton; Shariq, Jamal; Zaharias, Steve; Zhang, Jun

doi:10.7554/elife.84412

Cited by 5 publications

(9 citation statements)

References 78 publications

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“…Additionally, our findings emphasize the vital contribution of tyrosine residues in stabilizing Efg1 phase separation, as seen for other proteins including FUS (24, 32, 33). By disrupting these tyrosine-mediated interactions, we attenuated Efg1 phase separation, suggesting a central role for these residues in driving the process.…”

Section: Discussionsupporting

confidence: 73%

“…Drawing from previous studies (32,33) and our data that both highlighted the critical role of tyrosine residues in promoting phase separation, we then tested the role of tyrosine residues within both the helical and disordered regions of Efg1 N. We introduced mutations that replaced aromatic tyrosine with serine in these regions (34), and subsequently assessed their effects on phase separation (Fig. 6C,E).…”

Section: Tyrosine Residues In Helical Regions Stabilize Phase Separationmentioning

confidence: 99%

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Structure and position-specific interactions of prion-like domains in transcription factor Efg1 phase separation

Wang,

Zheng,

Fawzi

2023

Preprint

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Candida albicans, a prominent member of the human microbiome, can make an opportunistic switch from commensal coexistence to pathogenicity accompanied by an epigenetic shift between the white and opaque cell states. This transcriptional switch is under precise regulation by a set of transcription factors (TFs), with Enhanced Filamentous Growth Protein 1 (Efg1) playing a central role. Previous research has emphasized the importance of Egf1’s prion-like domain (PrLD) and the protein’s ability to undergo phase separation for the white-to-opaque transition ofC. albicans. However, the underlying molecular mechanisms of Efg1 phase separation have remained underexplored. In this study, we delved into the biophysical basis of Efg1 phase separation, revealing the significant contribution of both N-terminal (N) and C-terminal (C) PrLDs. Through NMR structural analysis, we found that Efg1 N-PrLD and C-PrLD are mostly disordered though have prominent partial α-helical secondary structures in both domains. NMR titration experiments suggest that the partially helical structures in N-PrLD act as hubs for self-interaction as well as Efg1 interaction with RNA. Using condensed-phase NMR spectroscopy, we uncovered diverse amino acid interactions underlying Efg1 phase separation. Particularly, we highlight the indispensable role of tyrosine residues within the transient α-helical structures of PrLDs particularly in the N-PrLD compared to the C-PrLD in stabilizing phase separation. Our study provides evidence that the transient α-helical structure is present in the phase separated state and highlights the particular importance of aromatic residues within these structures for phase separation. Together, these results enhance the understanding ofC. albicansTF interactions that lead to virulence and provide a crucial foundation for potential antifungal therapies targeting the transcriptional switch.Statement of SignificancePhase separated condensates have been found across the domains of life and many types of cells. To understand their varied functions, seeing the residue-by-residue details of the structure and interactions of component protein constituents is essential. A set of transcription factors that phase-separate controls cell fate of the pathogenic yeast Candida albicans. Here, we examine the structural and interaction details of a main regulator of this process, Efg1, using NMR spectroscopy and biochemical assays. We find Efg1’s phase-separating domains are not entirely disordered as often assumed but in fact contain helical regions that persist upon phase separation. We also reveal the balance of contacts formed in the condensed phase and the importance of specific residues and regions in phase separation.

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Section: Discussionsupporting

confidence: 73%

Section: Tyrosine Residues In Helical Regions Stabilize Phase Separationmentioning

confidence: 99%

Structure and position-specific interactions of prion-like domains in transcription factor Efg1 phase separation

Wang,

Zheng,

Fawzi

2023

Preprint

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“…Our recent study has revealed that SRSF1 undergoes phase separation in a physiological buffer (Fargason et l., 2023). We discovered that the interaction between the RS region and the conserved RRM sites, composed of acidic and aromatic residues, is responsible for the phase separation of SRSF1.…”

Section: Commentarymentioning

confidence: 99%

“…We discovered that the interaction between the RS region and the conserved RRM sites, composed of acidic and aromatic residues, is responsible for the phase separation of SRSF1. Given that these interacting regions are conserved throughout the SR protein family, phase separation appears to be a common feature in this protein family (Fargason et al., 2023).…”

Section: Commentarymentioning

confidence: 99%

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Purification of SRSF1 from E. coli for Biophysical and Biochemical Assays

Zhang,

Zhang

2024

Current Protocols

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The Ser/Arg‐rich splicing factors (SR proteins) constitute a crucial protein family in alternative splicing, comprising twelve members characterized by unique repetitive Arg‐Ser dipeptide sequences (RS) and one to two RNA‐recognition motifs (RRM). The RS regions of SR proteins undergo variable phosphorylation, resulting in unphosphorylated, partially phosphorylated, or hyper‐phosphorylated states based on functional requirements. Despite the identification of the SR protein family over 30 years ago, the purification of native SR proteins in soluble form at large quantities has presented challenges due to their low solubility. This protocol delineates a method for acquiring soluble, full‐length, unphosphorylated, hypo‐ and hyper‐phosphorylated SRSF1, a prototypical SR family member. Notably, this protocol facilitates the purification of SRSF1 in ample quantities suitable for NMR, as well as various biophysical and biochemical studies. The methodologies and principles outlined herein are expected to extend beyond SRSF1 protein production and can be adapted for purifying other SR protein family members or SR‐related proteins, such as snRNP70 and U2AF‐35. Given the involvement of these proteins in numerous essential biological processes, this protocol will prove beneficial to researchers in related fields. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Purification of SRSF1 from E. coliSupport Protocol: Purification of ULP1Basic Protocol 2: Purification of hypo‐phosphorylated SRSF1 from E. coliBasic Protocol 3: Purification of hyper‐phosphorylated SRSF1 from E. coli

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The U1‐70K and SRSF1 interaction is modulated by phosphorylation during the early stages of spliceosome assembly

Paul,

Zhang,

Zhang

et al. 2024

Protein Science

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In eukaryotes, pre‐mRNA splicing is vital for RNA processing and orchestrated by the spliceosome, whose assembly starts with the interaction between U1‐70K and SR proteins. Despite the significance of the U1‐70K/SR interaction, the dynamic nature of the complex and the challenges in obtaining soluble U1‐70K have impeded a comprehensive understanding of the interaction at the structural level for decades. We overcome the U1‐70K solubility issues, enabling us to characterize the interaction between U1‐70K and SRSF1, a representative SR protein. We unveil specific interactions: phosphorylated SRSF1 RS with U1‐70K BAD1, and SRSF1 RRM1 with U1‐70K RRM. The RS/BAD1 interaction plays a dominant role, whereas the interaction between the RRM domains further enhances the stability of the U1‐70K/SRSF1 complex. The RRM interaction involves the C‐terminal extension of U1‐70K RRM and the conserved acid patches on SRSF1 RRM1 that is involved in SRSF1 phase separation. Our circular dichroism spectra reveal that BAD1 adapts an α‐helical conformation and RS is intrinsically disordered. Intriguingly, BAD1 undergoes a conformation switch from α‐helix to β‐strand and random coil upon RS binding. In addition to the regulatory mechanism via SRSF1 phosphorylation, the U1‐70K/SRSF1 interaction is also regulated by U1‐70K BAD1 phosphorylation. We find that U1‐70K phosphorylation inhibits the U1‐70K and SRSF1 interaction. Our structural findings are validated through in vitro splicing assays and in‐cell saturated domain scanning using the CRISPR method, providing new insights into the intricate regulatory mechanisms of pre‐mRNA splicing.

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Peptides that Mimic RS repeats modulate phase separation of SRSF1, revealing a reliance on combined stacking and electrostatic interactions

Cited by 5 publications

References 78 publications

Structure and position-specific interactions of prion-like domains in transcription factor Efg1 phase separation

Structure and position-specific interactions of prion-like domains in transcription factor Efg1 phase separation

Purification of SRSF1 from E. coli for Biophysical and Biochemical Assays

The U1‐70K and SRSF1 interaction is modulated by phosphorylation during the early stages of spliceosome assembly

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