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The DExH box helicase domain of spindle-E is necessary for retrotransposon silencing and axial patterning during Drosophila oogenesis.

Ott KM, Nguyen T, Navarro C - G3 (Bethesda) (2014)

Bottom Line: Of the alleles that express detectable Spindle-E protein, we found that five had mutations in the DExH box domain.The phenotype of many of these alleles is as severe as the strongest spindle-E phenotype, whereas alleles with mutations in other regions of Spindle-E did not affect these processes as much.From these data we conclude that the DExH box domain of Spindle-E is necessary for its function in the piRNA pathway and retrotransposon silencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Biomedical Genetics, Boston University School of Medicine, Boston, Massachusetts 02118 Graduate Program in Genetics and Genomics, Boston University School of Medicine, Boston, Massachusetts 02118.

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Eight of the fourteen spn-E alleles express detectable protein and have point mutations in the SPN-E coding region. (A) Domain structure of Drosophila SPN-E and its human homolog, TDRD9. SPN-E contains a highly conserved DExH box and a Tudor domain as well as a Zinc finger, whereas TDRD9 only has a DExH box and Tudor domain. The position of the two mutations outside of the conserved domains, the five mutations that do not produce detectable protein, and the Zinc finger are shown. (B) The amino acid sequence of the SPN-E DExH box domain compared with its human homolog TDRD9, yeast splicing factor Prp16, and vaccinia virus protein NPH-I. The positions of the five mutations identified in the SPN-E DExH box domain are shown. Amino acid numbering is according to Ensemble Genome Browser release 73. (C) SPN-E protein expression in mutant ovary extracts as measured by Western blotting. Protein was isolated from hemizygous ovaries of the genotype spn-Emutant/spn-E∆125. Eight alleles express detectable protein of the correct size for SPN-E. Four alleles do not express detectable protein. Spn-E/Bal = spn-E∆125/Balancer chromosome. Line 7G2-5 is not shown. Several extraneous bands are found on the Western blots shown above. We did not detect these bands when we used a second antibody developed in the laboratory of Dr. Toshie Kai (Patil and Kai 2010; data not shown); therefore, we think that the extra bands are most likely nonspecific bands recognized by our SPN-E antibody. (D) SPN-E protein levels in the various mutant ovaries relative to spn-E∆125/Balancer. Error bars represent SD of at least 2 separate protein isolates. SPN-E protein levels were normalized to beta-tubulin. (E) A listing of each spn-E allele name along with its corresponding mutation.
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fig1: Eight of the fourteen spn-E alleles express detectable protein and have point mutations in the SPN-E coding region. (A) Domain structure of Drosophila SPN-E and its human homolog, TDRD9. SPN-E contains a highly conserved DExH box and a Tudor domain as well as a Zinc finger, whereas TDRD9 only has a DExH box and Tudor domain. The position of the two mutations outside of the conserved domains, the five mutations that do not produce detectable protein, and the Zinc finger are shown. (B) The amino acid sequence of the SPN-E DExH box domain compared with its human homolog TDRD9, yeast splicing factor Prp16, and vaccinia virus protein NPH-I. The positions of the five mutations identified in the SPN-E DExH box domain are shown. Amino acid numbering is according to Ensemble Genome Browser release 73. (C) SPN-E protein expression in mutant ovary extracts as measured by Western blotting. Protein was isolated from hemizygous ovaries of the genotype spn-Emutant/spn-E∆125. Eight alleles express detectable protein of the correct size for SPN-E. Four alleles do not express detectable protein. Spn-E/Bal = spn-E∆125/Balancer chromosome. Line 7G2-5 is not shown. Several extraneous bands are found on the Western blots shown above. We did not detect these bands when we used a second antibody developed in the laboratory of Dr. Toshie Kai (Patil and Kai 2010; data not shown); therefore, we think that the extra bands are most likely nonspecific bands recognized by our SPN-E antibody. (D) SPN-E protein levels in the various mutant ovaries relative to spn-E∆125/Balancer. Error bars represent SD of at least 2 separate protein isolates. SPN-E protein levels were normalized to beta-tubulin. (E) A listing of each spn-E allele name along with its corresponding mutation.

Mentions: A critical protein involved in the generation of most germ cell piRNA species is Drosophila Spindle-E (Malone et al. 2009). SPN-E colocalizes to the nuage along with other piRNA pathway proteins and its function is required for either primary piRNA generation and/or the ping-pong cycle (Malone et al. 2009; Patil and Kai 2010). spn-E was originally identified as a gene necessary for microtubule network formation, RNA localization, and embryonic pattern formation (Gillespie and Berg 1995; Klattenhoff et al. 2007; Martin et al. 2003). However, it is not known whether SPN-E function in the piRNA pathway controls all of these processes. The SPN-E protein contains a DExH box helicase domain, a Tudor domain, and a Zinc finger (Zn), which implicate its function in RNA processing, translational regulation, RNA decay, splicing, or protein–protein interactions (Figure 1, A and B). However, the relative contribution of these domains to SPN-E function, particularly in the piRNA pathway, is currently unknown. Therefore, to begin to understand how SPN-E functions during oogenesis, particularly in TE silencing, we took advantage of several previously isolated spn-E mutant fly lines in an attempt to identify mutations in the predicted functional domains. Our results provide evidence that the DExH box helicase domain of SPN-E is necessary for TE silencing in the germline.


The DExH box helicase domain of spindle-E is necessary for retrotransposon silencing and axial patterning during Drosophila oogenesis.

Ott KM, Nguyen T, Navarro C - G3 (Bethesda) (2014)

Eight of the fourteen spn-E alleles express detectable protein and have point mutations in the SPN-E coding region. (A) Domain structure of Drosophila SPN-E and its human homolog, TDRD9. SPN-E contains a highly conserved DExH box and a Tudor domain as well as a Zinc finger, whereas TDRD9 only has a DExH box and Tudor domain. The position of the two mutations outside of the conserved domains, the five mutations that do not produce detectable protein, and the Zinc finger are shown. (B) The amino acid sequence of the SPN-E DExH box domain compared with its human homolog TDRD9, yeast splicing factor Prp16, and vaccinia virus protein NPH-I. The positions of the five mutations identified in the SPN-E DExH box domain are shown. Amino acid numbering is according to Ensemble Genome Browser release 73. (C) SPN-E protein expression in mutant ovary extracts as measured by Western blotting. Protein was isolated from hemizygous ovaries of the genotype spn-Emutant/spn-E∆125. Eight alleles express detectable protein of the correct size for SPN-E. Four alleles do not express detectable protein. Spn-E/Bal = spn-E∆125/Balancer chromosome. Line 7G2-5 is not shown. Several extraneous bands are found on the Western blots shown above. We did not detect these bands when we used a second antibody developed in the laboratory of Dr. Toshie Kai (Patil and Kai 2010; data not shown); therefore, we think that the extra bands are most likely nonspecific bands recognized by our SPN-E antibody. (D) SPN-E protein levels in the various mutant ovaries relative to spn-E∆125/Balancer. Error bars represent SD of at least 2 separate protein isolates. SPN-E protein levels were normalized to beta-tubulin. (E) A listing of each spn-E allele name along with its corresponding mutation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: Eight of the fourteen spn-E alleles express detectable protein and have point mutations in the SPN-E coding region. (A) Domain structure of Drosophila SPN-E and its human homolog, TDRD9. SPN-E contains a highly conserved DExH box and a Tudor domain as well as a Zinc finger, whereas TDRD9 only has a DExH box and Tudor domain. The position of the two mutations outside of the conserved domains, the five mutations that do not produce detectable protein, and the Zinc finger are shown. (B) The amino acid sequence of the SPN-E DExH box domain compared with its human homolog TDRD9, yeast splicing factor Prp16, and vaccinia virus protein NPH-I. The positions of the five mutations identified in the SPN-E DExH box domain are shown. Amino acid numbering is according to Ensemble Genome Browser release 73. (C) SPN-E protein expression in mutant ovary extracts as measured by Western blotting. Protein was isolated from hemizygous ovaries of the genotype spn-Emutant/spn-E∆125. Eight alleles express detectable protein of the correct size for SPN-E. Four alleles do not express detectable protein. Spn-E/Bal = spn-E∆125/Balancer chromosome. Line 7G2-5 is not shown. Several extraneous bands are found on the Western blots shown above. We did not detect these bands when we used a second antibody developed in the laboratory of Dr. Toshie Kai (Patil and Kai 2010; data not shown); therefore, we think that the extra bands are most likely nonspecific bands recognized by our SPN-E antibody. (D) SPN-E protein levels in the various mutant ovaries relative to spn-E∆125/Balancer. Error bars represent SD of at least 2 separate protein isolates. SPN-E protein levels were normalized to beta-tubulin. (E) A listing of each spn-E allele name along with its corresponding mutation.
Mentions: A critical protein involved in the generation of most germ cell piRNA species is Drosophila Spindle-E (Malone et al. 2009). SPN-E colocalizes to the nuage along with other piRNA pathway proteins and its function is required for either primary piRNA generation and/or the ping-pong cycle (Malone et al. 2009; Patil and Kai 2010). spn-E was originally identified as a gene necessary for microtubule network formation, RNA localization, and embryonic pattern formation (Gillespie and Berg 1995; Klattenhoff et al. 2007; Martin et al. 2003). However, it is not known whether SPN-E function in the piRNA pathway controls all of these processes. The SPN-E protein contains a DExH box helicase domain, a Tudor domain, and a Zinc finger (Zn), which implicate its function in RNA processing, translational regulation, RNA decay, splicing, or protein–protein interactions (Figure 1, A and B). However, the relative contribution of these domains to SPN-E function, particularly in the piRNA pathway, is currently unknown. Therefore, to begin to understand how SPN-E functions during oogenesis, particularly in TE silencing, we took advantage of several previously isolated spn-E mutant fly lines in an attempt to identify mutations in the predicted functional domains. Our results provide evidence that the DExH box helicase domain of SPN-E is necessary for TE silencing in the germline.

Bottom Line: Of the alleles that express detectable Spindle-E protein, we found that five had mutations in the DExH box domain.The phenotype of many of these alleles is as severe as the strongest spindle-E phenotype, whereas alleles with mutations in other regions of Spindle-E did not affect these processes as much.From these data we conclude that the DExH box domain of Spindle-E is necessary for its function in the piRNA pathway and retrotransposon silencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Biomedical Genetics, Boston University School of Medicine, Boston, Massachusetts 02118 Graduate Program in Genetics and Genomics, Boston University School of Medicine, Boston, Massachusetts 02118.

Show MeSH
Related in: MedlinePlus