Arginine-Enriched Mixed-Charge Domains Provide Cohesion for Nuclear Speckle Condensation

Greig, Jamie A.; Nguyen, Tu Anh; Lee, Michelle; Holehouse, Alex S.; Posey, Ammon E.; Pappu, Rohit V.; Jedd, Gregory

doi:10.1016/j.molcel.2020.01.025

Cited by 163 publications

(205 citation statements)

References 109 publications

(129 reference statements)

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“…These polyampholytes resembles the mixed charge domains of nuclear speckle proteins, which contain repeats of positively and negatively charged residues. Increasing the fractions of arginine residues increases the propensity to condensate, which is consistent with the greater propensity for intramolecular selfassociation observed here (54). Intriguingly, this shows that the ratio between the 2 positively charged residues can tune the functional properties of IDPs in vivo.…”

Section: A B Csupporting

confidence: 88%

Effective concentrations enforced by intrinsically disordered linkers are governed by polymer physics

Sørensen

Kjærgaard

2019

Proc. Natl. Acad. Sci. U.S.A.

134

137

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Many multidomain proteins contain disordered linkers that regulate interdomain contacts, and thus the effective concentrations that govern intramolecular reactions. Effective concentrations are rarely measured experimentally, and therefore little is known about how they relate to linker architecture. We have directly measured the effective concentrations enforced by disordered protein linkers using a fluorescent biosensor. We show that effective concentrations follow simple geometric models based on polymer physics, offering an indirect method to probe the structural properties of the linker. The compaction of the disordered linker depends not only on net charge, but also on the type of charged residues. In contrast to theoretical predictions, we found that polyampholyte linkers can contract to similar dimensions as globular proteins. Hydrophobicity has little effect in itself, but aromatic residues lead to strong compaction, likely through π-interactions. Finally, we find that the individual contributors to chain compaction are not additive. We thus demonstrate that direct measurement of effective concentrations can be used in systematic studies of the relationship between sequence and structure of intrinsically disordered proteins. A quantitative understanding of the relationship between effective concentration and linker sequence will be crucial for understanding disorder-based allosteric regulation in multidomain proteins.

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Section: A B Csupporting

confidence: 88%

Effective concentrations enforced by intrinsically disordered linkers are governed by polymer physics

Sørensen

Kjærgaard

2019

Proc. Natl. Acad. Sci. U.S.A.

134

137

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“…Biomolecular condensates accommodate hundreds of distinct molecular components and considerable effort is being invested to understand the determinants of compositional control of distinct condensates (1, 9,15,17,19,34,36,[44][45][46][47][48][49][50]. Partition coefficients have become the workhorse quantities that are used to map the compositional biases of condensates (29,34,48,51).…”

Section: Discussionmentioning

confidence: 99%

Ligand Effects on Phase Separation of Multivalent Macromolecules

Ruff

Dar

Pappu

2020

Preprint

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Biomolecular condensates are thought to form via phase transitions of multivalent macromolecules. Components of condensates have been classified as scaffolds and clients. In this classification, scaffolds drive condensate formation whereas clients partition into condensates. However, non-scaffold molecules can be ligands that modulate the phase behavior of scaffolds via preferential binding across phase boundaries. Here, we present computational results that explain how preferential binding of ligands to scaffold molecules in different phases can impact phase separation and the compositions of dense phases. We show that phase separation is destabilized by monovalent ligands. In contrast, multivalent ligands, depending on their mode of binding, can stabilize phase separation by serving as crosslinkers that network scaffold molecules. We also find that concentrations of scaffolds within dense phases are generally diluted by ligands. This arises because ligands destabilize condensates by preferentially binding to scaffolds in the dilute phase or because ligands have to be accommodated within dense phases when they bind preferentially to scaffolds in the dense phase. Importantly, we show that partition coefficients, which are routinely used for compositional profiling of condensates, say nothing about the regulatory impact of ligands on the phase behavior of scaffolds. Therefore, instead of measuring partition coefficients it becomes imperative to measure how ligands impact threshold concentrations of scaffolds. These measurements, performed as a function of ligand concentration, will help with understanding the regulation of condensates and enable the design of molecules that impact condensate formation or dissolution.

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“…In order to test this hypothesis, we used the SRRM2 tr0 and SRRM2 tr10 cells as a model, which 280 allowed us to simultaneously detect SON, SRRM2 and an additional NS marker in the same cell. We chose SRRM1, which is used as a marker for NS in immunofluorescence experiments (Blencowe et al, 2000(Blencowe et al, , 1998(Blencowe et al, , 1994Rai et al, 2018;Zhang et al, 2016) and located at IGCs in electron microscopy experiments (Wan et al, 1994); RBM25, which is one of two recommended factors to mark NS by the Human Protein Atlas ("The human cell in nucleoplasm -The Human Protein Atlas," n.d.; Thul et al, 2017) (the other being SRRM2), localizes to NS through its RE/RD-rich mixed-charge domain (Zhou et al, 2008) that was 287 recently shown to target proteins to NS (Greig et al, 2020) and PNN, which localizes to nuclear speckles in human cells (Chiu and Ouyang, 2006;Joo et al, 2005;Lin et al, 2004;Zimowska et al, 2003). As reported previously (Ahn et al, 2011;Fei et al, 2017;Sharma et al, 2010), depletion of SON leads to collapsed speckles in SRRM2 tr0 cells, with SRRM2, SRRM1, PNN and RBM25 localizing to these spherical NS to different extents ( Figure 5A, Figure 5B, Figure 5 -figure supplement 3, compare SRRM2 tr0 cells, control vs SON siRNA treatment).…”

Section: Ns Formation Requires Son and Full-length Srrm2mentioning

confidence: 99%

SON and SRRM2 are essential for nuclear speckle formation

Ilık

Malszycki

Lübke

et al. 2020

eLife

155

173

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The nucleus of higher eukaryotes is a highly compartmentalized and dynamic organelle consisting of several biomolecular condensates that regulate gene expression at multiple levels (Banani et al., 2017; Shin and Brangwynne, 2017). First reported more than 100 years ago by Ramón y Cajal, nuclear speckles (NS) are among the most prominent of such condensates (Spector and Lamond, 2011). Despite their prevalence, research on the function of NS is virtually restricted to colocalization analyses, since an organizing core, without which NS cannot form, remains unidentified (Chen and Belmont, 2019; Galganski et al., 2017). The monoclonal antibody SC35, which was raised against a spliceosomal extract, is a frequently used reagent to mark NS since its debut in 1990 (Fu and Maniatis, 1990). Unexpectedly, we found that this antibody has been misidentified and the main target of SC35 mAb is SRRM2, a large (~300 kDa), spliceosome-associated (Jia and Sun, 2018) protein with prominent intrinsically disordered regions (IDRs) that sharply localizes to NS (Blencowe et al., 1994). Here we show that, the core of NS is likely formed by SON and SRRM2, since depletion of SON leads only to a partial disassembly of NS as reported previously (Ahn et al., 2011; Fei et al., 2017; Sharma et al., 2010), in contrast, combined depletion of SON together with SRRM2, but not other NS associated factors, or depletion of SON in a cell line where IDRs of SRRM2 are genetically deleted, leads to a near-complete dissolution of NS. This work, therefore, paves the way to study the role of NS under diverse physiological and stress conditions.

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Arginine-Enriched Mixed-Charge Domains Provide Cohesion for Nuclear Speckle Condensation

Cited by 163 publications

References 109 publications

Effective concentrations enforced by intrinsically disordered linkers are governed by polymer physics

Effective concentrations enforced by intrinsically disordered linkers are governed by polymer physics

Ligand Effects on Phase Separation of Multivalent Macromolecules

SON and SRRM2 are essential for nuclear speckle formation

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