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Nxf1 natural variant E610G is a semi-dominant suppressor of IAP-induced RNA processing defects.

Concepcion D, Ross KD, Hutt KR, Yeo GW, Hamilton BA - PLoS Genet. (2015)

Bottom Line: These insertions typically show more modest gene expression changes than de novo mutations, suggesting selection or attenuation.Genome-wide splicing-sensitive microarrays and gene-focused assays confirm specificity of Nxf1 genetic modifier activity for IAP insertion alleles.Strikingly, CRISPR/Cas9-mediated genome editing demonstrates that a single amino acid substitution in Nxf1, E610G, is sufficient to recreate a quantitative genetic modifier in a co-isogenic background.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Moores UCSD Cancer Center and Institute for Genomic Medicine, University of California, San Diego School of Medicine, La Jolla, California, United States of America; Department of Medicine, University of California, San Diego School of Medicine, La Jolla, California, United States of America.

ABSTRACT
Endogenous retroviruses and retrotransposons contribute functional genetic variation in animal genomes. In mice, Intracisternal A Particles (IAPs) are a frequent source of both new mutations and polymorphism across laboratory strains. Intronic IAPs can induce alternative RNA processing choices, including alternative splicing. We previously showed IAP I∆1 subfamily insertional mutations are suppressed by a wild-derived allele of the major mRNA export factor, Nxf1. Here we show that a wider diversity of IAP insertions present in the mouse reference sequence induce insertion-dependent alternative processing that is suppressed by Nxf1CAST alleles. These insertions typically show more modest gene expression changes than de novo mutations, suggesting selection or attenuation. Genome-wide splicing-sensitive microarrays and gene-focused assays confirm specificity of Nxf1 genetic modifier activity for IAP insertion alleles. Strikingly, CRISPR/Cas9-mediated genome editing demonstrates that a single amino acid substitution in Nxf1, E610G, is sufficient to recreate a quantitative genetic modifier in a co-isogenic background.

No MeSH data available.


Related in: MedlinePlus

E610G variant accounts for Modifier-of-vibrator activity of Nxf1.(A) The E610G edited allele decreased tremor severity. Tremor scores for vibrator mutant and littermate control animals of the indicated genotypes are plotted. Each point represents the mean observer score for one animal. P-values for the single hypothesis tests that one copy of the edited allele significantly reduced tremor relative to the unedited or pseudo-edited (ps.) B6 allele are shown. (B) The E610G edited allele increased genotype-dependent survival of vibrator animals. Kaplan-Meier plots show fraction alive over a ~3-month observation period. Heterozygotes for edited alleles are drawn in red, pseudo edited in blue. Censored animals are indicated with vertical lines. (C) Brain Pitpna gene expression from vibrator mutant alleles was increased by E610G alleles. Measurements were made from animals in (A) not used in (B). Relative quantities compared to three reference genes (Gapdh, Sdha, and Ppia) are plotted. Individual points represent the average of three replicate measurements per biological sample.
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pgen.1005123.g005: E610G variant accounts for Modifier-of-vibrator activity of Nxf1.(A) The E610G edited allele decreased tremor severity. Tremor scores for vibrator mutant and littermate control animals of the indicated genotypes are plotted. Each point represents the mean observer score for one animal. P-values for the single hypothesis tests that one copy of the edited allele significantly reduced tremor relative to the unedited or pseudo-edited (ps.) B6 allele are shown. (B) The E610G edited allele increased genotype-dependent survival of vibrator animals. Kaplan-Meier plots show fraction alive over a ~3-month observation period. Heterozygotes for edited alleles are drawn in red, pseudo edited in blue. Censored animals are indicated with vertical lines. (C) Brain Pitpna gene expression from vibrator mutant alleles was increased by E610G alleles. Measurements were made from animals in (A) not used in (B). Relative quantities compared to three reference genes (Gapdh, Sdha, and Ppia) are plotted. Individual points represent the average of three replicate measurements per biological sample.

Mentions: The hypothesis that the E610G polymorphism is sufficient for the original modifier phenotype makes three explicit predictions: that the edited allele should suppress vibrator phenotypes relative to the background Nxf1B6 (and silent, pseudo-edited) allele, that it should act semi-dominantly (and therefore be measurable in heterozygotes), and that it should be indistinguishable from the congenic Nxf1CAST allele in magnitude of effect. We examined 68 sequential vibrator (Pitpnvb/vb) mutant and 47 control G2 animals in backcrosses of our edited alleles to the B6–Pitpnavb/+, Nxf1B6/CAST stock (Fig. 5A). The E610G edited allele, but not the pseudo-edited control allele, altered the tremor phenotype of vibrator mutants–scored by observers blind to genotype–relative to the Nxf1B6 allele (S1_Video and S2_Video). This held for heterozygotes in trans to the Nxf1B6 allele (p = 7.9x10-6, Wilcoxon Rank Sum Test) and in trans to the Nxf1CAST allele (p = 2.0x10-4). The E610G edited allele was indistinguishable from the congenic Nxf1CAST allele in each context (Fig. 5A).


Nxf1 natural variant E610G is a semi-dominant suppressor of IAP-induced RNA processing defects.

Concepcion D, Ross KD, Hutt KR, Yeo GW, Hamilton BA - PLoS Genet. (2015)

E610G variant accounts for Modifier-of-vibrator activity of Nxf1.(A) The E610G edited allele decreased tremor severity. Tremor scores for vibrator mutant and littermate control animals of the indicated genotypes are plotted. Each point represents the mean observer score for one animal. P-values for the single hypothesis tests that one copy of the edited allele significantly reduced tremor relative to the unedited or pseudo-edited (ps.) B6 allele are shown. (B) The E610G edited allele increased genotype-dependent survival of vibrator animals. Kaplan-Meier plots show fraction alive over a ~3-month observation period. Heterozygotes for edited alleles are drawn in red, pseudo edited in blue. Censored animals are indicated with vertical lines. (C) Brain Pitpna gene expression from vibrator mutant alleles was increased by E610G alleles. Measurements were made from animals in (A) not used in (B). Relative quantities compared to three reference genes (Gapdh, Sdha, and Ppia) are plotted. Individual points represent the average of three replicate measurements per biological sample.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383553&req=5

pgen.1005123.g005: E610G variant accounts for Modifier-of-vibrator activity of Nxf1.(A) The E610G edited allele decreased tremor severity. Tremor scores for vibrator mutant and littermate control animals of the indicated genotypes are plotted. Each point represents the mean observer score for one animal. P-values for the single hypothesis tests that one copy of the edited allele significantly reduced tremor relative to the unedited or pseudo-edited (ps.) B6 allele are shown. (B) The E610G edited allele increased genotype-dependent survival of vibrator animals. Kaplan-Meier plots show fraction alive over a ~3-month observation period. Heterozygotes for edited alleles are drawn in red, pseudo edited in blue. Censored animals are indicated with vertical lines. (C) Brain Pitpna gene expression from vibrator mutant alleles was increased by E610G alleles. Measurements were made from animals in (A) not used in (B). Relative quantities compared to three reference genes (Gapdh, Sdha, and Ppia) are plotted. Individual points represent the average of three replicate measurements per biological sample.
Mentions: The hypothesis that the E610G polymorphism is sufficient for the original modifier phenotype makes three explicit predictions: that the edited allele should suppress vibrator phenotypes relative to the background Nxf1B6 (and silent, pseudo-edited) allele, that it should act semi-dominantly (and therefore be measurable in heterozygotes), and that it should be indistinguishable from the congenic Nxf1CAST allele in magnitude of effect. We examined 68 sequential vibrator (Pitpnvb/vb) mutant and 47 control G2 animals in backcrosses of our edited alleles to the B6–Pitpnavb/+, Nxf1B6/CAST stock (Fig. 5A). The E610G edited allele, but not the pseudo-edited control allele, altered the tremor phenotype of vibrator mutants–scored by observers blind to genotype–relative to the Nxf1B6 allele (S1_Video and S2_Video). This held for heterozygotes in trans to the Nxf1B6 allele (p = 7.9x10-6, Wilcoxon Rank Sum Test) and in trans to the Nxf1CAST allele (p = 2.0x10-4). The E610G edited allele was indistinguishable from the congenic Nxf1CAST allele in each context (Fig. 5A).

Bottom Line: These insertions typically show more modest gene expression changes than de novo mutations, suggesting selection or attenuation.Genome-wide splicing-sensitive microarrays and gene-focused assays confirm specificity of Nxf1 genetic modifier activity for IAP insertion alleles.Strikingly, CRISPR/Cas9-mediated genome editing demonstrates that a single amino acid substitution in Nxf1, E610G, is sufficient to recreate a quantitative genetic modifier in a co-isogenic background.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Moores UCSD Cancer Center and Institute for Genomic Medicine, University of California, San Diego School of Medicine, La Jolla, California, United States of America; Department of Medicine, University of California, San Diego School of Medicine, La Jolla, California, United States of America.

ABSTRACT
Endogenous retroviruses and retrotransposons contribute functional genetic variation in animal genomes. In mice, Intracisternal A Particles (IAPs) are a frequent source of both new mutations and polymorphism across laboratory strains. Intronic IAPs can induce alternative RNA processing choices, including alternative splicing. We previously showed IAP I∆1 subfamily insertional mutations are suppressed by a wild-derived allele of the major mRNA export factor, Nxf1. Here we show that a wider diversity of IAP insertions present in the mouse reference sequence induce insertion-dependent alternative processing that is suppressed by Nxf1CAST alleles. These insertions typically show more modest gene expression changes than de novo mutations, suggesting selection or attenuation. Genome-wide splicing-sensitive microarrays and gene-focused assays confirm specificity of Nxf1 genetic modifier activity for IAP insertion alleles. Strikingly, CRISPR/Cas9-mediated genome editing demonstrates that a single amino acid substitution in Nxf1, E610G, is sufficient to recreate a quantitative genetic modifier in a co-isogenic background.

No MeSH data available.


Related in: MedlinePlus