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Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles.

Nott TJ, Petsalaki E, Farber P, Jervis D, Fussner E, Plochowietz A, Craggs TD, Bazett-Jones DP, Pawson T, Forman-Kay JD, Baldwin AJ - Mol. Cell (2015)

Bottom Line: These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temperature, ionic strength, arginine methylation, and splicing.Moreover, the bodies provide an alternative solvent environment that can concentrate single-stranded DNA but largely exclude double-stranded DNA.We propose that phase separation of disordered proteins containing weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles.

View Article: PubMed Central - PubMed

Affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada; Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ, UK.

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The Sequence Features that Enable Droplet Formation by Ddx4 and Their Distribution within the Human Genome(A) Sliding net charge (10 amino acid window, black) is shown for (i) Ddx4N1 and (ii) a charge-scrambled mutant, Ddx4N1CS, obtained by swapping the positions of positive residues (blue bars) and negative residues (red bars) to minimize any persistence of blocks of charge. (iii) A mutant where nine phenylalanine residues, whose placement was highly conserved, were mutated to alanine (Ddx4N1FtoA, see Figure S6). The positions of the nine phenylalanine residues (yellow circles) mutated to alanine are indicated.(B) Representative fluorescence images from cell imaging experiments reveal that Ddx4N1CS and Ddx4N1FtoA do not form organelles in cells under physiological conditions. Residual HeLa nucleoli are still observed as fluorescence-depleted regions within the cell nucleus.(C) The human genome was surveyed for sequences with similar physical properties to the Ddx4 disordered termini. 1,556 sequences out of 14,198 were identified to have [F/R]G spacings in their sequence that are similar to the Ddx4 ortholog family. The top 10% of these are indicated (dotted line). A significant number of proteins associated with forming non-membrane organelles were present in this group.(D) Similar plots from the yeast (i) and E. coli (ii) genomes revealing a number of proteins closely associated with nucleic acid biochemistry.
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fig6: The Sequence Features that Enable Droplet Formation by Ddx4 and Their Distribution within the Human Genome(A) Sliding net charge (10 amino acid window, black) is shown for (i) Ddx4N1 and (ii) a charge-scrambled mutant, Ddx4N1CS, obtained by swapping the positions of positive residues (blue bars) and negative residues (red bars) to minimize any persistence of blocks of charge. (iii) A mutant where nine phenylalanine residues, whose placement was highly conserved, were mutated to alanine (Ddx4N1FtoA, see Figure S6). The positions of the nine phenylalanine residues (yellow circles) mutated to alanine are indicated.(B) Representative fluorescence images from cell imaging experiments reveal that Ddx4N1CS and Ddx4N1FtoA do not form organelles in cells under physiological conditions. Residual HeLa nucleoli are still observed as fluorescence-depleted regions within the cell nucleus.(C) The human genome was surveyed for sequences with similar physical properties to the Ddx4 disordered termini. 1,556 sequences out of 14,198 were identified to have [F/R]G spacings in their sequence that are similar to the Ddx4 ortholog family. The top 10% of these are indicated (dotted line). A significant number of proteins associated with forming non-membrane organelles were present in this group.(D) Similar plots from the yeast (i) and E. coli (ii) genomes revealing a number of proteins closely associated with nucleic acid biochemistry.

Mentions: A notable feature of Ddx4 disordered termini is that they arrange their charged residues into clustered blocks of net positive and negative charge (Figure 6Ai), resembling a block co-polymer. The clusters persist for approximately 8–10 residues in length and tend to contain 3–8 similarly charged residues. To determine the physical importance of this charge patterning, we produced a Ddx4 variant, Ddx4N1CS, with the same overall net charge, but in which the blocks were scrambled (Figure S6G). In Ddx4N1CS, the regions of opposing charge are removed while simultaneously maintaining the same overall isoelectric point (PI), amino acid composition, and positions of all other residues (Figure 6Aii). This construct was unable to form organelles in vitro under near-physiological conditions. When expressed in cells in the charge-scrambled form of Ddx4YFP, the protein accumulated to high concentrations without forming organelle-like structures (Figure 6Bii), revealing the importance of charge patterning in organelle formation.


Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles.

Nott TJ, Petsalaki E, Farber P, Jervis D, Fussner E, Plochowietz A, Craggs TD, Bazett-Jones DP, Pawson T, Forman-Kay JD, Baldwin AJ - Mol. Cell (2015)

The Sequence Features that Enable Droplet Formation by Ddx4 and Their Distribution within the Human Genome(A) Sliding net charge (10 amino acid window, black) is shown for (i) Ddx4N1 and (ii) a charge-scrambled mutant, Ddx4N1CS, obtained by swapping the positions of positive residues (blue bars) and negative residues (red bars) to minimize any persistence of blocks of charge. (iii) A mutant where nine phenylalanine residues, whose placement was highly conserved, were mutated to alanine (Ddx4N1FtoA, see Figure S6). The positions of the nine phenylalanine residues (yellow circles) mutated to alanine are indicated.(B) Representative fluorescence images from cell imaging experiments reveal that Ddx4N1CS and Ddx4N1FtoA do not form organelles in cells under physiological conditions. Residual HeLa nucleoli are still observed as fluorescence-depleted regions within the cell nucleus.(C) The human genome was surveyed for sequences with similar physical properties to the Ddx4 disordered termini. 1,556 sequences out of 14,198 were identified to have [F/R]G spacings in their sequence that are similar to the Ddx4 ortholog family. The top 10% of these are indicated (dotted line). A significant number of proteins associated with forming non-membrane organelles were present in this group.(D) Similar plots from the yeast (i) and E. coli (ii) genomes revealing a number of proteins closely associated with nucleic acid biochemistry.
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fig6: The Sequence Features that Enable Droplet Formation by Ddx4 and Their Distribution within the Human Genome(A) Sliding net charge (10 amino acid window, black) is shown for (i) Ddx4N1 and (ii) a charge-scrambled mutant, Ddx4N1CS, obtained by swapping the positions of positive residues (blue bars) and negative residues (red bars) to minimize any persistence of blocks of charge. (iii) A mutant where nine phenylalanine residues, whose placement was highly conserved, were mutated to alanine (Ddx4N1FtoA, see Figure S6). The positions of the nine phenylalanine residues (yellow circles) mutated to alanine are indicated.(B) Representative fluorescence images from cell imaging experiments reveal that Ddx4N1CS and Ddx4N1FtoA do not form organelles in cells under physiological conditions. Residual HeLa nucleoli are still observed as fluorescence-depleted regions within the cell nucleus.(C) The human genome was surveyed for sequences with similar physical properties to the Ddx4 disordered termini. 1,556 sequences out of 14,198 were identified to have [F/R]G spacings in their sequence that are similar to the Ddx4 ortholog family. The top 10% of these are indicated (dotted line). A significant number of proteins associated with forming non-membrane organelles were present in this group.(D) Similar plots from the yeast (i) and E. coli (ii) genomes revealing a number of proteins closely associated with nucleic acid biochemistry.
Mentions: A notable feature of Ddx4 disordered termini is that they arrange their charged residues into clustered blocks of net positive and negative charge (Figure 6Ai), resembling a block co-polymer. The clusters persist for approximately 8–10 residues in length and tend to contain 3–8 similarly charged residues. To determine the physical importance of this charge patterning, we produced a Ddx4 variant, Ddx4N1CS, with the same overall net charge, but in which the blocks were scrambled (Figure S6G). In Ddx4N1CS, the regions of opposing charge are removed while simultaneously maintaining the same overall isoelectric point (PI), amino acid composition, and positions of all other residues (Figure 6Aii). This construct was unable to form organelles in vitro under near-physiological conditions. When expressed in cells in the charge-scrambled form of Ddx4YFP, the protein accumulated to high concentrations without forming organelle-like structures (Figure 6Bii), revealing the importance of charge patterning in organelle formation.

Bottom Line: These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temperature, ionic strength, arginine methylation, and splicing.Moreover, the bodies provide an alternative solvent environment that can concentrate single-stranded DNA but largely exclude double-stranded DNA.We propose that phase separation of disordered proteins containing weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles.

View Article: PubMed Central - PubMed

Affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada; Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ, UK.

Show MeSH
Related in: MedlinePlus