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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

Nxf1 E610G genome editing in mouse embryos.(A) Translation and genomic sequence surrounding E610G targeted for editing. Codon 610 is shaded grey, targeted bases are in red and boxed, guide RNA sequence is in blue, the presumed Cas9 cut site shown by arrowheads, and the PAM sequence is underlined. (B) Partial sequence of the repair template oligonucleotide shows the two targeted base changes, including one in the guide RNA sequence. (C) Sequences obtained by PCR from tail DNA of one dead (Pup 2) and two live (Pups 3 and 4) G0 pups. Sequence from cloned PCR products (Pups 2 and 3) confirmed an equivalent 3-bp deletion (ΔVal) on one allele in each animal. Sequence from offspring confirmed transmission of each edited allele from the two viable founders.
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pgen.1005123.g004: Nxf1 E610G genome editing in mouse embryos.(A) Translation and genomic sequence surrounding E610G targeted for editing. Codon 610 is shaded grey, targeted bases are in red and boxed, guide RNA sequence is in blue, the presumed Cas9 cut site shown by arrowheads, and the PAM sequence is underlined. (B) Partial sequence of the repair template oligonucleotide shows the two targeted base changes, including one in the guide RNA sequence. (C) Sequences obtained by PCR from tail DNA of one dead (Pup 2) and two live (Pups 3 and 4) G0 pups. Sequence from cloned PCR products (Pups 2 and 3) confirmed an equivalent 3-bp deletion (ΔVal) on one allele in each animal. Sequence from offspring confirmed transmission of each edited allele from the two viable founders.

Mentions: The precise genetic variant or variants responsible for the modifier activity of Nxf1 have not been previously determined. As Nxf1 steady-state RNA and protein levels did not appear different between alleles, we reasoned that one or both of two amino acid substitutions that differentiate the alleles was likely causal. Because the E610G variant occurred in a highly-conserved domain and appeared to be the last variant that arose in a 19-marker haplotype before that haplotype rose to high frequency in wild mice [20], we targeted this site for genome editing in mouse one cell embryos, using a synthetic Cas9 mRNA and a single guide RNA (Fig. 4A) co-injected with an oligonucleotide template for homology-dependent repair (Fig. 4B), essentially as described [40]. Two potential founders survived. Each survivor was male and heterozygous for two distinctly edited alleles–(1) a correctly edited allele containing both the E610G polymorphism and a silent polymorphism in the sgRNA homology to deter further cleavage, and (2) either a “pseudo-edited” allele, carrying the induced silent polymorphism, or the E610G edit together with a 3-bp deletion of the adjacent valine codon (Fig. 4C). Both males bred and transmitted each allele. All four transmitted alleles were detected by PCR sequencing tail-clip DNA from the founders and their offspring. Sequencing of 17 predicted [41] off-target cleavage sites from the same samples identified no additional mutations, suggesting that off-target editing was not frequent in either founder.


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)

Nxf1 E610G genome editing in mouse embryos.(A) Translation and genomic sequence surrounding E610G targeted for editing. Codon 610 is shaded grey, targeted bases are in red and boxed, guide RNA sequence is in blue, the presumed Cas9 cut site shown by arrowheads, and the PAM sequence is underlined. (B) Partial sequence of the repair template oligonucleotide shows the two targeted base changes, including one in the guide RNA sequence. (C) Sequences obtained by PCR from tail DNA of one dead (Pup 2) and two live (Pups 3 and 4) G0 pups. Sequence from cloned PCR products (Pups 2 and 3) confirmed an equivalent 3-bp deletion (ΔVal) on one allele in each animal. Sequence from offspring confirmed transmission of each edited allele from the two viable founders.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4383553&req=5

pgen.1005123.g004: Nxf1 E610G genome editing in mouse embryos.(A) Translation and genomic sequence surrounding E610G targeted for editing. Codon 610 is shaded grey, targeted bases are in red and boxed, guide RNA sequence is in blue, the presumed Cas9 cut site shown by arrowheads, and the PAM sequence is underlined. (B) Partial sequence of the repair template oligonucleotide shows the two targeted base changes, including one in the guide RNA sequence. (C) Sequences obtained by PCR from tail DNA of one dead (Pup 2) and two live (Pups 3 and 4) G0 pups. Sequence from cloned PCR products (Pups 2 and 3) confirmed an equivalent 3-bp deletion (ΔVal) on one allele in each animal. Sequence from offspring confirmed transmission of each edited allele from the two viable founders.
Mentions: The precise genetic variant or variants responsible for the modifier activity of Nxf1 have not been previously determined. As Nxf1 steady-state RNA and protein levels did not appear different between alleles, we reasoned that one or both of two amino acid substitutions that differentiate the alleles was likely causal. Because the E610G variant occurred in a highly-conserved domain and appeared to be the last variant that arose in a 19-marker haplotype before that haplotype rose to high frequency in wild mice [20], we targeted this site for genome editing in mouse one cell embryos, using a synthetic Cas9 mRNA and a single guide RNA (Fig. 4A) co-injected with an oligonucleotide template for homology-dependent repair (Fig. 4B), essentially as described [40]. Two potential founders survived. Each survivor was male and heterozygous for two distinctly edited alleles–(1) a correctly edited allele containing both the E610G polymorphism and a silent polymorphism in the sgRNA homology to deter further cleavage, and (2) either a “pseudo-edited” allele, carrying the induced silent polymorphism, or the E610G edit together with a 3-bp deletion of the adjacent valine codon (Fig. 4C). Both males bred and transmitted each allele. All four transmitted alleles were detected by PCR sequencing tail-clip DNA from the founders and their offspring. Sequencing of 17 predicted [41] off-target cleavage sites from the same samples identified no additional mutations, suggesting that off-target editing was not frequent in either founder.

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