Regulation of RNA granule dynamics by phosphorylation of serine-rich, intrinsically disordered proteins in C. elegans

Wang, Jennifer T.; Smith, Jarrett; Chen, Bi-Chang; Schmidt, Helen; Rasoloson, Dominique; Paix, Alexandre; Lambrus, Bramwell G.; Calidas, Deepika; Betzig, Eric; Seydoux, Géraldine

doi:10.7554/elife.04591

Cited by 348 publications

(487 citation statements)

References 59 publications

Supporting

Mentioning

453

Contrasting

Order By: Relevance

“…However, it is estimated that as many as 30% (39) of proteins in the human genome have regions of intrinsic disorder, and IDPs appear to be involved in a range of biological functions, owing to their flexible conformations and binding promiscuity. Our findings are consistent with the emerging role of LCS/IDPs in promoting the assembly of RNP bodies (15,16,(40)(41)(42).…”

Section: Discussionsupporting

confidence: 80%

“…7). This picture is consistent with recent work suggesting a role in P granule assembly for two predicted IDP-containing proteins: MEG-1 and MEG-3 (41). The interactions between these proteins is controlled by phosphorylation, reflecting a balance between activity of the kinase MBK-2/DYRK and the phosphatase PPTR-1/2; this manifests in altered propensity for assembly of P granules.…”

Section: Discussionsupporting

confidence: 79%

See 1 more Smart Citation

The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

Elbaum‐Garfinkle

Kim

Szczepaniak

et al. 2015

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

1,104

1,292

View full text Add to dashboard Cite

P granules and other RNA/protein bodies are membrane-less organelles that may assemble by intracellular phase separation, similar to the condensation of water vapor into droplets. However, the molecular driving forces and the nature of the condensed phases remain poorly understood. Here, we show that the Caenorhabditis elegans protein LAF-1, a DDX3 RNA helicase found in P granules, phase separates into P granule-like droplets in vitro. We adapt a microrheology technique to precisely measure the viscoelasticity of micrometer-sized LAF-1 droplets, revealing purely viscous properties highly tunable by salt and RNA concentration. RNA decreases viscosity and increases molecular dynamics within the droplet. Single molecule FRET assays suggest that this RNA fluidization results from highly dynamic RNA-protein interactions that emerge close to the droplet phase boundary. We demonstrate than an N-terminal, arginine/glycine rich, intrinsically disordered protein (IDP) domain of LAF-1 is necessary and sufficient for both phase separation and RNA-protein interactions. In vivo, RNAi knockdown of LAF-1 results in the dissolution of P granules in the early embryo, with an apparent submicromolar phase boundary comparable to that measured in vitro. Together, these findings demonstrate that LAF-1 is important for promoting P granule assembly and provide insight into the mechanism by which IDP-driven molecular interactions give rise to liquid phase organelles with tunable properties.liquid droplets | intracellular phase transition | intrinsically disordered proteins | RNA granules I ntracellular RNA/protein (RNP) assemblies, including germ granules, processing bodies, stress granules, and nucleoli, are key players in the regulation of gene expression (1). RNP bodies, also referred to as RNA granules, function in diverse modes of RNA processing, including splicing, degradation, and translational repression of mRNA. These ubiquitous structures lack a membrane boundary but nonetheless represent a coherent organelle composed of thousands of molecules, manifesting as microscopically visible puncta in both the cytoplasm and the nucleus.Recent studies have demonstrated the apparent liquid-like behavior of various RNP bodies (2-5) including wetting, dripping, and relaxation to spherical structures upon fusion or shearing. The assembly and disassembly of liquid-like organelles appears to be governed by a phase separation process, demonstrated by a concentration-dependent condensation/dissolution of P granules (2, 6) and the assembly and size scaling of the nucleolus (7) in the Caenorhabditis elegans embryo. Liquid phase separation has also been suggested to play a role in stress granule assembly (4) and in multivalent signaling proteins (8). These studies lend increasing support to the hypothesis that liquid phases play a central role in intracellular organization. However, the specific molecular interactions that drive phase separation and the mechanisms by which liquid properties impart cellular function remain largely unclear. P granule...

show abstract

Section: Discussionsupporting

confidence: 80%

Section: Discussionsupporting

confidence: 79%

The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

Elbaum‐Garfinkle

Kim

Szczepaniak

et al. 2015

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

1,104

1,292

View full text Add to dashboard Cite

show abstract

“…Ribonucleoprotein (RNP) granules, such as germ-line P granules and the miRISC-resident P bodies in somatic tissues, possess hydrogel-like properties (70,71) whose dynamics of formation and dissolution depend, in part, on the phosphorylation of granule components. RNA binding proteins within these granules typically contain low complexity sequences at their N and C termini that form disordered domains (72).…”

Section: Discussionmentioning

confidence: 99%

Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1

Alessi

Khivansara

Han

et al. 2015

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

View full text Add to dashboard Cite

MicroRNAs (miRNAs) play essential, conserved roles in diverse developmental processes through association with the miRNA-induced silencing complex (miRISC). Whereas fundamental insights into the mechanistic framework of miRNA biogenesis and target gene silencing have been established, posttranslational modifications that affect miRISC function are less well understood. Here we report that the conserved serine/threonine kinase, casein kinase II (CK2), promotes miRISC function in Caenorhabditis elegans. CK2 inactivation results in developmental defects that phenocopy loss of miRISC cofactors and enhances the loss of miRNA function in diverse cellular contexts. Whereas CK2 is dispensable for miRNA biogenesis and the stability of miRISC cofactors, it is required for efficient miRISC target mRNA binding and silencing. Importantly, we identify the conserved DEAD-box RNA helicase, CGH-1/DDX6, as a key CK2 substrate within miRISC and demonstrate phosphorylation of a conserved N-terminal serine is required for CGH-1 function in the miRNA pathway.

show abstract

“…For example, methylation of RG/RGG repeats suppresses phase separation of the highly charged domains of nuage protein DDX4 (Nott et al , 2015). Phosphorylation of the low‐complexity domains of maternal‐effect germline defective (MEG) proteins 1 and 3 promotes processing granule disassembly in Caenorhabditis elegans embryos (Wang et al , 2014). Therefore, some low‐complexity modifications may alter LLPS by disrupting the interactions of negative/positive charged residue patterns (Nott et al , 2015; Lee et al , 2016).…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of the FUS low‐complexity domain disrupts phase separation, aggregation, and toxicity

et al. 2017

View full text Add to dashboard Cite

Neuronal inclusions of aggregated RNA‐binding protein fused in sarcoma (FUS) are hallmarks of ALS and frontotemporal dementia subtypes. Intriguingly, FUS's nearly uncharged, aggregation‐prone, yeast prion‐like, low sequence‐complexity domain (LC) is known to be targeted for phosphorylation. Here we map in vitro and in‐cell phosphorylation sites across FUS LC. We show that both phosphorylation and phosphomimetic variants reduce its aggregation‐prone/prion‐like character, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Nuclear magnetic resonance spectroscopy demonstrates the intrinsically disordered structure of FUS LC is preserved after phosphorylation; however, transient domain collapse and self‐interaction are reduced by phosphomimetics. Moreover, we show that phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS‐associated cytotoxicity. Hence, post‐translational modification may be a mechanism by which cells control physiological assembly and prevent pathological protein aggregation, suggesting a potential treatment pathway amenable to pharmacologic modulation.

show abstract

Regulation of RNA granule dynamics by phosphorylation of serine-rich, intrinsically disordered proteins in C. elegans

Cited by 348 publications

References 59 publications

The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1

Phosphorylation of the FUS low‐complexity domain disrupts phase separation, aggregation, and toxicity

Contact Info

Product

Resources

About