Limits...
A yeast model for polyalanine-expansion aggregation and toxicity.

Konopka CA, Locke MN, Gallagher PS, Pham N, Hart MP, Walker CJ, Gitler AD, Gardner RG - Mol. Biol. Cell (2011)

Bottom Line: Nine human disorders result from the toxic accumulation and aggregation of proteins with expansions in their endogenous polyalanine (polyA) tracts.In our initial case, we expanded the polyA tract within the native yeast poly(Adenine)-binding protein Pab1 from 8A to 13A, 15A, 17A, and 20A.Surprisingly, neither manipulation suppressed the cytotoxicity of 20A-expanded Pab1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, USA.

ABSTRACT
Nine human disorders result from the toxic accumulation and aggregation of proteins with expansions in their endogenous polyalanine (polyA) tracts. Given the prevalence of polyA tracts in eukaryotic proteomes, we wanted to understand the generality of polyA-expansion cytotoxicity by using yeast as a model organism. In our initial case, we expanded the polyA tract within the native yeast poly(Adenine)-binding protein Pab1 from 8A to 13A, 15A, 17A, and 20A. These expansions resulted in increasing formation of Pab1 inclusions, insolubility, and cytotoxicity that correlated with the length of the polyA expansion. Pab1 binds mRNA as part of its normal function, and disrupting RNA binding or altering cytoplasmic mRNA levels suppressed the cytotoxicity of 17A-expanded Pab1, indicating a requisite role for mRNA in Pab1 polyA-expansion toxicity. Surprisingly, neither manipulation suppressed the cytotoxicity of 20A-expanded Pab1. Thus longer expansions may have a different mechanism for toxicity. We think that this difference underscores the potential need to examine the cytotoxic mechanisms of both long and short expansions in models of expansion disorders.

Show MeSH

Related in: MedlinePlus

Pab117A but not Pab120A requires RNA binding for toxicity and aggregation. (A) Schematic of Pab1 domain structure. C, C-terminal domain; PR, proline rich. (B) Tenfold serial dilutions of cells were spotted onto medium containing glucose (expression repressed) or galactose (expression induced) to measure spotting efficiency and toxicity of Pab117A and Pab120A RNA-binding mutants. (C) Cells expressing RNA-binding mutants of Pab117A were assayed for presence of GFP-positive inclusions and morphology defects after 16-h induction and reported as a percentage of GFP-positive cells. More than 200 cells in three independent cultures were counted for each. Bars are SD. *p < 0.05 by Student's t test when compared with wild-type Pab117A.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3113764&req=5

Figure 8: Pab117A but not Pab120A requires RNA binding for toxicity and aggregation. (A) Schematic of Pab1 domain structure. C, C-terminal domain; PR, proline rich. (B) Tenfold serial dilutions of cells were spotted onto medium containing glucose (expression repressed) or galactose (expression induced) to measure spotting efficiency and toxicity of Pab117A and Pab120A RNA-binding mutants. (C) Cells expressing RNA-binding mutants of Pab117A were assayed for presence of GFP-positive inclusions and morphology defects after 16-h induction and reported as a percentage of GFP-positive cells. More than 200 cells in three independent cultures were counted for each. Bars are SD. *p < 0.05 by Student's t test when compared with wild-type Pab117A.

Mentions: RNA binding has been shown to be a necessary feature of toxicity for several disease-associated mutations in RNA-binding proteins (Fan et al., 2001; Tavanez et al., 2005; Chartier et al., 2006; Johnson et al., 2008; Voigt et al., 2010). Therefore we examined whether this was also the case for polyA-expanded Pab1. Pab1 contains four RNA recognition motifs (RRMs): RRM1, RRM2, RRM3, and RRM4 (Sachs et al., 1986, and Figure 8A). RRM1 and RRM2 have poly(Ade)-binding capability (Burd et al., 1991), and are involved in 3′ mRNA poly(Ade)-tail binding, with RRM2 contributing the majority of affinity (Deardorff and Sachs, 1997). RRM3 and RRM4 possess nonspecific RNA-binding capability (Burd et al., 1991; Deardorff and Sachs, 1997), with RRM4 contributing most of the RNA-binding capability (Deardorff and Sachs, 1997). The RRM domain typically comprises two conserved motifs termed RNP1 and RNP2 (Maris et al., 2005). Within each motif, a single aromatic residue Phe/Tyr is necessary for RNA base-stacking interactions (Maris et al., 2005). Substitution of this residue with a nonaromatic residue (Leu or Val) reduces the ability of RRM-containing RNA-binding proteins to bind RNA (Deardorff and Sachs, 1997).


A yeast model for polyalanine-expansion aggregation and toxicity.

Konopka CA, Locke MN, Gallagher PS, Pham N, Hart MP, Walker CJ, Gitler AD, Gardner RG - Mol. Biol. Cell (2011)

Pab117A but not Pab120A requires RNA binding for toxicity and aggregation. (A) Schematic of Pab1 domain structure. C, C-terminal domain; PR, proline rich. (B) Tenfold serial dilutions of cells were spotted onto medium containing glucose (expression repressed) or galactose (expression induced) to measure spotting efficiency and toxicity of Pab117A and Pab120A RNA-binding mutants. (C) Cells expressing RNA-binding mutants of Pab117A were assayed for presence of GFP-positive inclusions and morphology defects after 16-h induction and reported as a percentage of GFP-positive cells. More than 200 cells in three independent cultures were counted for each. Bars are SD. *p < 0.05 by Student's t test when compared with wild-type Pab117A.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113764&req=5

Figure 8: Pab117A but not Pab120A requires RNA binding for toxicity and aggregation. (A) Schematic of Pab1 domain structure. C, C-terminal domain; PR, proline rich. (B) Tenfold serial dilutions of cells were spotted onto medium containing glucose (expression repressed) or galactose (expression induced) to measure spotting efficiency and toxicity of Pab117A and Pab120A RNA-binding mutants. (C) Cells expressing RNA-binding mutants of Pab117A were assayed for presence of GFP-positive inclusions and morphology defects after 16-h induction and reported as a percentage of GFP-positive cells. More than 200 cells in three independent cultures were counted for each. Bars are SD. *p < 0.05 by Student's t test when compared with wild-type Pab117A.
Mentions: RNA binding has been shown to be a necessary feature of toxicity for several disease-associated mutations in RNA-binding proteins (Fan et al., 2001; Tavanez et al., 2005; Chartier et al., 2006; Johnson et al., 2008; Voigt et al., 2010). Therefore we examined whether this was also the case for polyA-expanded Pab1. Pab1 contains four RNA recognition motifs (RRMs): RRM1, RRM2, RRM3, and RRM4 (Sachs et al., 1986, and Figure 8A). RRM1 and RRM2 have poly(Ade)-binding capability (Burd et al., 1991), and are involved in 3′ mRNA poly(Ade)-tail binding, with RRM2 contributing the majority of affinity (Deardorff and Sachs, 1997). RRM3 and RRM4 possess nonspecific RNA-binding capability (Burd et al., 1991; Deardorff and Sachs, 1997), with RRM4 contributing most of the RNA-binding capability (Deardorff and Sachs, 1997). The RRM domain typically comprises two conserved motifs termed RNP1 and RNP2 (Maris et al., 2005). Within each motif, a single aromatic residue Phe/Tyr is necessary for RNA base-stacking interactions (Maris et al., 2005). Substitution of this residue with a nonaromatic residue (Leu or Val) reduces the ability of RRM-containing RNA-binding proteins to bind RNA (Deardorff and Sachs, 1997).

Bottom Line: Nine human disorders result from the toxic accumulation and aggregation of proteins with expansions in their endogenous polyalanine (polyA) tracts.In our initial case, we expanded the polyA tract within the native yeast poly(Adenine)-binding protein Pab1 from 8A to 13A, 15A, 17A, and 20A.Surprisingly, neither manipulation suppressed the cytotoxicity of 20A-expanded Pab1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, USA.

ABSTRACT
Nine human disorders result from the toxic accumulation and aggregation of proteins with expansions in their endogenous polyalanine (polyA) tracts. Given the prevalence of polyA tracts in eukaryotic proteomes, we wanted to understand the generality of polyA-expansion cytotoxicity by using yeast as a model organism. In our initial case, we expanded the polyA tract within the native yeast poly(Adenine)-binding protein Pab1 from 8A to 13A, 15A, 17A, and 20A. These expansions resulted in increasing formation of Pab1 inclusions, insolubility, and cytotoxicity that correlated with the length of the polyA expansion. Pab1 binds mRNA as part of its normal function, and disrupting RNA binding or altering cytoplasmic mRNA levels suppressed the cytotoxicity of 17A-expanded Pab1, indicating a requisite role for mRNA in Pab1 polyA-expansion toxicity. Surprisingly, neither manipulation suppressed the cytotoxicity of 20A-expanded Pab1. Thus longer expansions may have a different mechanism for toxicity. We think that this difference underscores the potential need to examine the cytotoxic mechanisms of both long and short expansions in models of expansion disorders.

Show MeSH
Related in: MedlinePlus