Sequence-Specific Polyampholyte Phase Separation in Membraneless Organelles

Lin, Yi‐Hsuan; Forman-Kay, Julie D.; Chan, Hue Sun

doi:10.1103/physrevlett.117.178101

Cited by 243 publications

(362 citation statements)

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“…Nuclear bodies often assemble through the binding of RBPs containing intrinsically disordered regions to scaffolding RNAs, and their oligomerization induces a liquid-liquid phase separation 31 . To assess whether SRSF7 bodies are phase-separated nuclear bodies, we treated SRSF7 OE cells with 10% 1,6-HD, which disrupts weak hydrophobic interactions [32][33][34] , and performed RNA FISH. Indeed, 1,6-HD treatment efficiently dispersed SRSF7 bodies into numerous smaller foci that still colocalized with SRSF7-GFP.…”

Section: Intronmentioning

confidence: 99%

SRSF7 maintains its homeostasis through the expression of Split-ORFs and nuclear body assembly

Königs

Machado

Arnold

et al. 2020

Nat Struct Mol Biol

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Results SRSF7 overexpression induces auto-regulation.To investigate the mechanisms of SRSF7 homeostasis, we generated cell lines SRSF7 is an essential RNA-binding protein whose misexpression promotes cancer. Here, we describe how SRSF7 maintains its protein homeostasis in murine P19 cells using an intricate negative feedback mechanism. SRSF7 binding to its premessenger RNA promotes inclusion of a poison cassette exon and transcript degradation via nonsense-mediated decay (NMD). However, elevated SRSF7 levels inhibit NMD and promote translation of two protein halves, termed Split-ORFs, from the bicistronic SRSF7-PCE transcript. The first half acts as dominant-negative isoform suppressing poison cassette exon inclusion and instead promoting the retention of flanking introns containing repeated SRSF7 binding sites. Massive SRSF7 binding to these sites and its oligomerization promote the assembly of large nuclear bodies, which sequester SRSF7 transcripts at their transcription site, preventing their export and restoring normal SRSF7 protein levels. We further show that hundreds of human and mouse NMD targets, especially RNA-binding proteins, encode potential Split-ORFs, some of which are expressed under specific cellular conditions. SRSF7 binding promotes splicing of NMD-sensitive and -resistant SRSF7 isoforms. To understand the mechanism of SRSF7 auto-regulation, we examined the binding of SRSF7 protein to SRSF7 transcripts using individual-nucleotide resolution ultraviolet (UV) crosslinking and immunoprecipitation (iCLIP). We used normalized significant crosslink events (X-links, false discovery rate (FDR) < 0.05) from SRSF3 and SRSF7 iCLIP datasets of P19 cell lines without OE 8 . Similar to SRSF3, which promotes the inclusion of the PCE in SRSF7 transcripts 17 , SRSF7 showed enriched crosslinks in an extended region encompassing the PCE, its flanking introns 3a and 3b, and exons 3, 4 and 5 ( Fig. 1d). Quantification revealed that SRSF7 binds ~50-fold more to SRSF7 transcripts than SRSF3 ( Supplementary Table 1), indicating that SRSF7 has an unusual preference for its own transcripts.RNA-seq followed by quantification of junction reads revealed that SRSF7 OE promotes inclusion of either the complete PCE (460 nucleotides (nt)) or a partial PCE (107 nt) in SRSF7 transcripts ( Fig. 1e). Additionally, both PCE-flanking introns (3a and 3b) and intron 5 displayed increased read coverage, indicating that they are partly retained upon SRSF7 OE. Semiquantitative reverse transcription PCR and sequencing confirmed that SRSF7 OE caused the appearance of transcripts containing the entire PCE (SRSF7-PCE, orange asterisk), the partial PCE (SRSF7-PCE 1/4 , yellow asterisk) or the PCE in combination with both flanking introns (SRSF7-I3a+b, red asterisk) or with only intron 3b (SRSF7-I3b, green asterisk; Fig. 1f and Extended Data Fig. 1b,c). Identical isoforms were detected for endogenous and SRSF7-GFP reporter transcripts, indicating that auto-regulation operates similarly on both.All these transcripts contain PTCs and should be s...

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Section: Intronmentioning

confidence: 99%

SRSF7 maintains its homeostasis through the expression of Split-ORFs and nuclear body assembly

Königs

Machado

Arnold

et al. 2020

Nat Struct Mol Biol

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“…Ddx4’s sequence is arranged in 8–10 residue blocks of alternating charge density, and this patterning is intrinsic to Ddx4’s behavior: a scrambled construct of identical composition was unable to phase separate under physiological conditions, despite accumulating to high concentrations in cells . In addition, arginine methylation and phenylalanine mutation or disruption both abrogated droplet formation (Figure ) …”

Section: Three Examples: Proteins Mlos and Functionmentioning

confidence: 99%

“…Together, this suggests π–cation interactions between the positively charged arginine and the aromatic phenylalanine contribute to the electrostatic force that allows Ddx4 to condense from bulk solution. Furthermore, Ddx4 constructs in which all arginines were mutated to lysines, which preserve the positive charge but lack a π system, failed to phase separate, suggesting a specific role for arginine in phase separation through π–π interactions . The efficacy of these interactions at driving phase separation is apparently dependent on their sequence position within the alternating charge blocks …”

Section: Three Examples: Proteins Mlos and Functionmentioning

confidence: 99%

“…Although electrostatic forces are the primary driver of Ddx4’s phase separation, theoretical calculations showed that theirs is not the only contribution. Electrostatics‐only models failed to match the experimental phase diagrams for Ddx4, and the equations only recapitulated the data once aromatic interactions were considered . The free energy contribution from entropy was also calculated to favor droplet formation under all conditions, but the contribution was comparatively small, allowing droplets to condense and dissolve primarily according to electrostatic tuning …”

Section: Three Examples: Proteins Mlos and Functionmentioning

confidence: 99%

“…Ddx4 highlights an increasing understanding of the role that amino acid sequence, not just composition, plays in phase separation. Developments in random‐phase approximation theory describe this role in terms of electrostatic interactions, finding that phase‐separation propensity is directly dependent on the number of alternating charge blocks in a protein . Unlike Flory–Huggins or Overbeek–Voorn theories, random‐phase approximation theory accounts for residue sequence and sequence‐correlated interactions, allowing the theory to recapitulate the experimental result that charge‐scrambled Ddx4 fails to phase separate.…”

Section: Three Examples: Proteins Mlos and Functionmentioning

confidence: 99%

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Physical Chemistry of Cellular Liquid‐Phase Separation

Bentley

Frey

Deniz

2019

Chemistry A European J

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Compartmentalization of biochemical processes is essential for cell function. Although membrane‐bound organelles are well studied in this context, recent work has shown that phase separation is a key contributor to cellular compartmentalization through the formation of liquid‐like membraneless organelles (MLOs). In this Minireview, the key mechanistic concepts that underlie MLO dynamics and function are first briefly discussed, including the relevant noncovalent interaction chemistry and polymer physical chemistry. Next, a few examples of MLOs and relevant proteins are given, along with their functions, which highlight the relevance of the above concepts. The developing area of active matter and non‐equilibrium systems, which can give rise to unexpected effects in fluctuating cellular conditions, are also discussed. Finally, our thoughts for emerging and future directions in the field are discussed, including in vitro and in vivo studies of MLO physical chemistry and function.

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Future directions in physiochemical modeling of the thermodynamics of polyelectrolyte coacervates

Ghasemi

Larson

2022

AIChE Journal

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We review theories of polyelectrolyte (PE) coacervation, which is the spontaneous association of oppositely charged units of PEs and phase separation into a polymerdense phase in aqueous solution. The simplest theories can be divided into "physicsbased" and "chemistry-based" approaches. In the former, PEs are treated as charged, long-chain, molecules, defined by charge level, chain length, and chain flexibility, but otherwise lacking chemical identity, with electrostatic interactions driving coacervation. The "chemistry-based" approaches focus on the local interactions between the species for which chemical identity is critical, and describe coacervation as the result of competitive local binding interactions of monomers and salts. In this article, we show how these approaches complement each other by presenting recent approaches that take both physical and chemical effects into account. Finally, we suggest future directions toward producing theories that are made quantitatively predictive by accounting for both long range electrostatic and local chemically specific interactions.

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Sequence-Specific Polyampholyte Phase Separation in Membraneless Organelles

Cited by 243 publications

References 48 publications

SRSF7 maintains its homeostasis through the expression of Split-ORFs and nuclear body assembly

SRSF7 maintains its homeostasis through the expression of Split-ORFs and nuclear body assembly

Physical Chemistry of Cellular Liquid‐Phase Separation

Future directions in physiochemical modeling of the thermodynamics of polyelectrolyte coacervates

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