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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

Adamts13S is suppressed by congenic Nxf1CAST.(A) Alignment of Adamts13 protein domains to their corresponding exons in Adamts13L and Adamts13S shows loss of two thrombospondin (TSP) and two CUB domains from Adamts13S, which terminates in an intronic IAP sequence. TM, transmembrane segment; PRO, protease domain. Locations of primers to detect exon 24 to exon 25 splicing around the IAP are indicated. (B) The Adamts13S IAP shares greater sequence similarity with the AtrnmgL full-length IAP than with IΔ1 elements, an example of which is shown. Percent nucleotide identity is shown for indicated segments. (C) Quantitative RT-PCR for exon 24-exon 25 splice junction relative to Gapdh in B6 x BALB/c F2 mice homozygous for both Adamts13S and the indicated congenic allele of Nxf1 shows substantial increase in the Nxf1CAST congenic allele (p = 6.4x10-5, Wilcoxon rank sum test). (D) Similar results were obtained for a smaller number of animals from the B6 congenic line (p = 1.1x10-3, Wilcoxon rank sum test). Normalization to Desmin as a cell type-specific marker increased the separation of values. Each dot in the box plots represents the mean of technical replicates for one biological sample.
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pgen.1005123.g001: Adamts13S is suppressed by congenic Nxf1CAST.(A) Alignment of Adamts13 protein domains to their corresponding exons in Adamts13L and Adamts13S shows loss of two thrombospondin (TSP) and two CUB domains from Adamts13S, which terminates in an intronic IAP sequence. TM, transmembrane segment; PRO, protease domain. Locations of primers to detect exon 24 to exon 25 splicing around the IAP are indicated. (B) The Adamts13S IAP shares greater sequence similarity with the AtrnmgL full-length IAP than with IΔ1 elements, an example of which is shown. Percent nucleotide identity is shown for indicated segments. (C) Quantitative RT-PCR for exon 24-exon 25 splice junction relative to Gapdh in B6 x BALB/c F2 mice homozygous for both Adamts13S and the indicated congenic allele of Nxf1 shows substantial increase in the Nxf1CAST congenic allele (p = 6.4x10-5, Wilcoxon rank sum test). (D) Similar results were obtained for a smaller number of animals from the B6 congenic line (p = 1.1x10-3, Wilcoxon rank sum test). Normalization to Desmin as a cell type-specific marker increased the separation of values. Each dot in the box plots represents the mean of technical replicates for one biological sample.

Mentions: Because congenic Nxf1CAST showed modifier activity against several insertions of the IAP IΔ1 subfamily, but not against a single example of a full-length element [19, 20], we tested its activity on a well-studied IAP allele of a different class (Fig. 1). Adamts13 encodes a large circulating protease responsible for processing multimeric von Willebrand factor (vWF) in vivo [22, 23]; mutations in human ADAMTS13 cause thrombotic thrombocytopenic purpura [24] and variations in ADAMTS13 activity are associated with other thrombotic abnormalities [25, 26]. In the Adamts13S allele present in several inbred strains, an IAP insertion into intron 24 results in an alternatively spliced, stable transcript that terminates in viral sequences, but lacks downstream exons encoding the final two thrombospondin repeats and two CUB (C1r/C1s, Uegf, Bmp1) domains [27]. A very small amount of residual exon 24 to exon 25 splicing can be detected by a quantitative reverse transcription–polymerase chain reaction (qRT-PCR) assays (Ref. 27 and Fig. 1A). The Adamts13S IAP sequence is highly similar to the AtrnmgL IAP (Fig. 1B), but differs in having a 1131-bp deletion in the pol coding region (Fig. 1B), distinct from and smaller than the classical IΔ2 deletion [21]. Of 2329 bp among 107 aligned sites that distinguish the AtrnmgL IAP from the IΔ1 elements, only 33 sites (61 bp) are not shared between AtrnmgL and Adamts13S IAPs.


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)

Adamts13S is suppressed by congenic Nxf1CAST.(A) Alignment of Adamts13 protein domains to their corresponding exons in Adamts13L and Adamts13S shows loss of two thrombospondin (TSP) and two CUB domains from Adamts13S, which terminates in an intronic IAP sequence. TM, transmembrane segment; PRO, protease domain. Locations of primers to detect exon 24 to exon 25 splicing around the IAP are indicated. (B) The Adamts13S IAP shares greater sequence similarity with the AtrnmgL full-length IAP than with IΔ1 elements, an example of which is shown. Percent nucleotide identity is shown for indicated segments. (C) Quantitative RT-PCR for exon 24-exon 25 splice junction relative to Gapdh in B6 x BALB/c F2 mice homozygous for both Adamts13S and the indicated congenic allele of Nxf1 shows substantial increase in the Nxf1CAST congenic allele (p = 6.4x10-5, Wilcoxon rank sum test). (D) Similar results were obtained for a smaller number of animals from the B6 congenic line (p = 1.1x10-3, Wilcoxon rank sum test). Normalization to Desmin as a cell type-specific marker increased the separation of values. Each dot in the box plots represents the mean of technical replicates for one biological sample.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4383553&req=5

pgen.1005123.g001: Adamts13S is suppressed by congenic Nxf1CAST.(A) Alignment of Adamts13 protein domains to their corresponding exons in Adamts13L and Adamts13S shows loss of two thrombospondin (TSP) and two CUB domains from Adamts13S, which terminates in an intronic IAP sequence. TM, transmembrane segment; PRO, protease domain. Locations of primers to detect exon 24 to exon 25 splicing around the IAP are indicated. (B) The Adamts13S IAP shares greater sequence similarity with the AtrnmgL full-length IAP than with IΔ1 elements, an example of which is shown. Percent nucleotide identity is shown for indicated segments. (C) Quantitative RT-PCR for exon 24-exon 25 splice junction relative to Gapdh in B6 x BALB/c F2 mice homozygous for both Adamts13S and the indicated congenic allele of Nxf1 shows substantial increase in the Nxf1CAST congenic allele (p = 6.4x10-5, Wilcoxon rank sum test). (D) Similar results were obtained for a smaller number of animals from the B6 congenic line (p = 1.1x10-3, Wilcoxon rank sum test). Normalization to Desmin as a cell type-specific marker increased the separation of values. Each dot in the box plots represents the mean of technical replicates for one biological sample.
Mentions: Because congenic Nxf1CAST showed modifier activity against several insertions of the IAP IΔ1 subfamily, but not against a single example of a full-length element [19, 20], we tested its activity on a well-studied IAP allele of a different class (Fig. 1). Adamts13 encodes a large circulating protease responsible for processing multimeric von Willebrand factor (vWF) in vivo [22, 23]; mutations in human ADAMTS13 cause thrombotic thrombocytopenic purpura [24] and variations in ADAMTS13 activity are associated with other thrombotic abnormalities [25, 26]. In the Adamts13S allele present in several inbred strains, an IAP insertion into intron 24 results in an alternatively spliced, stable transcript that terminates in viral sequences, but lacks downstream exons encoding the final two thrombospondin repeats and two CUB (C1r/C1s, Uegf, Bmp1) domains [27]. A very small amount of residual exon 24 to exon 25 splicing can be detected by a quantitative reverse transcription–polymerase chain reaction (qRT-PCR) assays (Ref. 27 and Fig. 1A). The Adamts13S IAP sequence is highly similar to the AtrnmgL IAP (Fig. 1B), but differs in having a 1131-bp deletion in the pol coding region (Fig. 1B), distinct from and smaller than the classical IΔ2 deletion [21]. Of 2329 bp among 107 aligned sites that distinguish the AtrnmgL IAP from the IΔ1 elements, only 33 sites (61 bp) are not shared between AtrnmgL and Adamts13S IAPs.

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