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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.


E610G replicates the modifier effect on IAP insertions in the B6 genome.(A) Relative expression levels of Adamts13 and Nsdhl RNAs with the IAP-containing intron correctly spliced were measured by qRT-PCR from livers of animals heterozygous for the indicated Nxf1 congenic (B, Nxf1B6; C, Nxf1CAST) or E610G edited allele. Expression measures are relative to five reference genes (Desmin, Gapdh, Sdha, Ppia, Pitpna) and divided by average relative value. Individual points represent the average of three replicate measurements per biological sample. (B) Similar measurements from brain cDNA of the same animals for the indicated genes. Measures are relative to four reference genes (Gapdh, Sdha, Ppia, Pitpna). P values are shown for a one-tailed Wilcoxon-Mann-Whitney test for the predicted increase in E610G edited animals relative to B6 homozygotes.
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pgen.1005123.g006: E610G replicates the modifier effect on IAP insertions in the B6 genome.(A) Relative expression levels of Adamts13 and Nsdhl RNAs with the IAP-containing intron correctly spliced were measured by qRT-PCR from livers of animals heterozygous for the indicated Nxf1 congenic (B, Nxf1B6; C, Nxf1CAST) or E610G edited allele. Expression measures are relative to five reference genes (Desmin, Gapdh, Sdha, Ppia, Pitpna) and divided by average relative value. Individual points represent the average of three replicate measurements per biological sample. (B) Similar measurements from brain cDNA of the same animals for the indicated genes. Measures are relative to four reference genes (Gapdh, Sdha, Ppia, Pitpna). P values are shown for a one-tailed Wilcoxon-Mann-Whitney test for the predicted increase in E610G edited animals relative to B6 homozygotes.

Mentions: The discovery of B6-endogenous targets of the modifier activity in our original congenic stock (Figs. 1 and 2) predicts that if E610G amino acid substitution allele explains the modifier activity, it should act on expression level of those genes as well. We tested this prediction with 12 qRT-PCR assays on F1 samples that were heterozygous for the edited allele, but without potentially confounding Pitpnvb mutations (Fig. 6). Both Adamts13 and Nsdhl showed suppression by the edited allele equivalent to the congenic allele in liver, where both are well-expressed (Fig. 6A). In brain cDNA, six of seven genes that were suppressed by the congenic stock (Agbl4, Agtr1a, Cdh19, L3mbtl4, Pla2g4e, and Slc15a2) showed effects of the heterozygous edited allele, while the seventh (Fhit) did not approach independent significance in the heterozygote samples (Fig. 6B). In addition, two of three genes for which the congenic experiments showed a trend but did not provide significant support (Sntg1 and Dph5) also showed significant evidence of suppression by the edited allele (Fig. 6B). Each event survived correction for false discovery rate below or near q = 0.05 (S1 Table). The tested sites, including elements closely related to the non-suppressed insertion in AtrnmgL, other full-length elements, IΔ1 elements, and other deletion classes, were significantly increased in a semi-dominant manner by the edited allele. Alternative processing events on chromosome 19 that differed between congenic stocks (Fig. 3C,D) were not significantly different between edited and unedited alleles (S4 Fig), confirming that they represented strain variations in each gene rather than effects of the modifier locus. Together, these measurements confirm that the E610G variant is able to suppress IAP-related gene expression changes at the wide array of sites detected for the congenic allele by our genome-wide screen and explains the modifier effect of previous congenic and transgenic strains [19, 20].


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 replicates the modifier effect on IAP insertions in the B6 genome.(A) Relative expression levels of Adamts13 and Nsdhl RNAs with the IAP-containing intron correctly spliced were measured by qRT-PCR from livers of animals heterozygous for the indicated Nxf1 congenic (B, Nxf1B6; C, Nxf1CAST) or E610G edited allele. Expression measures are relative to five reference genes (Desmin, Gapdh, Sdha, Ppia, Pitpna) and divided by average relative value. Individual points represent the average of three replicate measurements per biological sample. (B) Similar measurements from brain cDNA of the same animals for the indicated genes. Measures are relative to four reference genes (Gapdh, Sdha, Ppia, Pitpna). P values are shown for a one-tailed Wilcoxon-Mann-Whitney test for the predicted increase in E610G edited animals relative to B6 homozygotes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4383553&req=5

pgen.1005123.g006: E610G replicates the modifier effect on IAP insertions in the B6 genome.(A) Relative expression levels of Adamts13 and Nsdhl RNAs with the IAP-containing intron correctly spliced were measured by qRT-PCR from livers of animals heterozygous for the indicated Nxf1 congenic (B, Nxf1B6; C, Nxf1CAST) or E610G edited allele. Expression measures are relative to five reference genes (Desmin, Gapdh, Sdha, Ppia, Pitpna) and divided by average relative value. Individual points represent the average of three replicate measurements per biological sample. (B) Similar measurements from brain cDNA of the same animals for the indicated genes. Measures are relative to four reference genes (Gapdh, Sdha, Ppia, Pitpna). P values are shown for a one-tailed Wilcoxon-Mann-Whitney test for the predicted increase in E610G edited animals relative to B6 homozygotes.
Mentions: The discovery of B6-endogenous targets of the modifier activity in our original congenic stock (Figs. 1 and 2) predicts that if E610G amino acid substitution allele explains the modifier activity, it should act on expression level of those genes as well. We tested this prediction with 12 qRT-PCR assays on F1 samples that were heterozygous for the edited allele, but without potentially confounding Pitpnvb mutations (Fig. 6). Both Adamts13 and Nsdhl showed suppression by the edited allele equivalent to the congenic allele in liver, where both are well-expressed (Fig. 6A). In brain cDNA, six of seven genes that were suppressed by the congenic stock (Agbl4, Agtr1a, Cdh19, L3mbtl4, Pla2g4e, and Slc15a2) showed effects of the heterozygous edited allele, while the seventh (Fhit) did not approach independent significance in the heterozygote samples (Fig. 6B). In addition, two of three genes for which the congenic experiments showed a trend but did not provide significant support (Sntg1 and Dph5) also showed significant evidence of suppression by the edited allele (Fig. 6B). Each event survived correction for false discovery rate below or near q = 0.05 (S1 Table). The tested sites, including elements closely related to the non-suppressed insertion in AtrnmgL, other full-length elements, IΔ1 elements, and other deletion classes, were significantly increased in a semi-dominant manner by the edited allele. Alternative processing events on chromosome 19 that differed between congenic stocks (Fig. 3C,D) were not significantly different between edited and unedited alleles (S4 Fig), confirming that they represented strain variations in each gene rather than effects of the modifier locus. Together, these measurements confirm that the E610G variant is able to suppress IAP-related gene expression changes at the wide array of sites detected for the congenic allele by our genome-wide screen and explains the modifier effect of previous congenic and transgenic strains [19, 20].

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.