<i>COX4-like</i>, a Nuclear-Encoded Mitochondrial Gene Duplicate, Is Essential for Male Fertility in <i>Drosophila melanogaster</i>
Recent studies on nuclear-encoded mitochondrial genes (N-mt genes) in <i>Drosophila melanogaster</i> have shown a unique pattern of expression for newly duplicated N-mt genes, with many duplicates having a testis-biased expression and playing an essential role in spermatogenesis. In this...
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MDPI AG
2022-02-01
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| Series: | Genes |
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| Online Access: | https://www.mdpi.com/2073-4425/13/3/424 |
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| author | Mohammadmehdi Eslamieh Ayda Mirsalehi Dragomira N. Markova Esther Betrán |
| author_facet | Mohammadmehdi Eslamieh Ayda Mirsalehi Dragomira N. Markova Esther Betrán |
| author_sort | Mohammadmehdi Eslamieh |
| collection | DOAJ |
| description | Recent studies on nuclear-encoded mitochondrial genes (N-mt genes) in <i>Drosophila melanogaster</i> have shown a unique pattern of expression for newly duplicated N-mt genes, with many duplicates having a testis-biased expression and playing an essential role in spermatogenesis. In this study, we investigated a newly duplicated N-mt gene—i.e., <i>Cytochrome c oxidase 4-like</i> (<i>COX4L</i>)—in order to understand its function and, consequently, the reason behind its retention in the <i>D. melanogaster</i> genome. The <i>COX4L</i> gene is a duplicate of the <i>Cytochrome c oxidase 4</i> (<i>COX4</i>) gene of OXPHOS complex IV. While the parental <i>COX4</i> gene has been found in all eukaryotes, including single-cell eukaryotes such as yeast, we show that <i>COX4L</i> is only present in the Brachycera suborder of Diptera; thus, both genes are present in all <i>Drosophila</i> species, but have significantly different patterns of expression: <i>COX4</i> is highly expressed in all tissues, while <i>COX4L</i> has a testis-specific expression. To understand the function of this new gene, we first knocked down its expression in the <i>D. melanogaster</i> germline using two different RNAi lines driven by the <i>bam-Gal4</i> driver; second, we created a knockout strain for this gene using CRISPR-Cas9 technology. Our results showed that knockdown and knockout lines of <i>COX4L</i> produce partial sterility and complete sterility in males, respectively, where a lack of sperm individualization was observed in both cases. Male infertility was prevented by driving <i>COX4L</i>-HA in the germline, but not when driving <i>COX4</i>-HA. In addition, ectopic expression of <i>COX4L</i> in the soma caused embryonic lethality, while overexpression in the germline led to a reduction in male fertility. <i>COX4L</i>-KO mitochondria show reduced membrane potential, providing a plausible explanation for the male sterility observed in these flies. This prominent loss-of-function phenotype, along with its testis-biased expression and its presence in the <i>Drosophila</i> sperm proteome, suggests that <i>COX4L</i> is a paralogous, specialized gene that is assembled in OXPHOS complex IV of male germline cells and/or sperm mitochondria. |
| first_indexed | 2024-03-09T19:48:40Z |
| format | Article |
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| institution | Directory Open Access Journal |
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| language | English |
| last_indexed | 2024-03-09T19:48:40Z |
| publishDate | 2022-02-01 |
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| spelling | doaj.art-cbd598a3ae084bcc9a5302a38c78b6392023-11-24T01:18:00ZengMDPI AGGenes2073-44252022-02-0113342410.3390/genes13030424<i>COX4-like</i>, a Nuclear-Encoded Mitochondrial Gene Duplicate, Is Essential for Male Fertility in <i>Drosophila melanogaster</i>Mohammadmehdi Eslamieh0Ayda Mirsalehi1Dragomira N. Markova2Esther Betrán3Department of Biology, The University of Texas at Arlington, Arlington, TX 76019, USADepartment of Biology, The University of Texas at Arlington, Arlington, TX 76019, USADepartment of Biology, The University of Texas at Arlington, Arlington, TX 76019, USADepartment of Biology, The University of Texas at Arlington, Arlington, TX 76019, USARecent studies on nuclear-encoded mitochondrial genes (N-mt genes) in <i>Drosophila melanogaster</i> have shown a unique pattern of expression for newly duplicated N-mt genes, with many duplicates having a testis-biased expression and playing an essential role in spermatogenesis. In this study, we investigated a newly duplicated N-mt gene—i.e., <i>Cytochrome c oxidase 4-like</i> (<i>COX4L</i>)—in order to understand its function and, consequently, the reason behind its retention in the <i>D. melanogaster</i> genome. The <i>COX4L</i> gene is a duplicate of the <i>Cytochrome c oxidase 4</i> (<i>COX4</i>) gene of OXPHOS complex IV. While the parental <i>COX4</i> gene has been found in all eukaryotes, including single-cell eukaryotes such as yeast, we show that <i>COX4L</i> is only present in the Brachycera suborder of Diptera; thus, both genes are present in all <i>Drosophila</i> species, but have significantly different patterns of expression: <i>COX4</i> is highly expressed in all tissues, while <i>COX4L</i> has a testis-specific expression. To understand the function of this new gene, we first knocked down its expression in the <i>D. melanogaster</i> germline using two different RNAi lines driven by the <i>bam-Gal4</i> driver; second, we created a knockout strain for this gene using CRISPR-Cas9 technology. Our results showed that knockdown and knockout lines of <i>COX4L</i> produce partial sterility and complete sterility in males, respectively, where a lack of sperm individualization was observed in both cases. Male infertility was prevented by driving <i>COX4L</i>-HA in the germline, but not when driving <i>COX4</i>-HA. In addition, ectopic expression of <i>COX4L</i> in the soma caused embryonic lethality, while overexpression in the germline led to a reduction in male fertility. <i>COX4L</i>-KO mitochondria show reduced membrane potential, providing a plausible explanation for the male sterility observed in these flies. This prominent loss-of-function phenotype, along with its testis-biased expression and its presence in the <i>Drosophila</i> sperm proteome, suggests that <i>COX4L</i> is a paralogous, specialized gene that is assembled in OXPHOS complex IV of male germline cells and/or sperm mitochondria.https://www.mdpi.com/2073-4425/13/3/424nuclear-encoded mitochondrial genegene duplicationCRISPR knockout<i>COX4L</i>spermatogenesis<i>Drosophila melanogaster</i> |
| spellingShingle | Mohammadmehdi Eslamieh Ayda Mirsalehi Dragomira N. Markova Esther Betrán <i>COX4-like</i>, a Nuclear-Encoded Mitochondrial Gene Duplicate, Is Essential for Male Fertility in <i>Drosophila melanogaster</i> Genes nuclear-encoded mitochondrial gene gene duplication CRISPR knockout <i>COX4L</i> spermatogenesis <i>Drosophila melanogaster</i> |
| title | <i>COX4-like</i>, a Nuclear-Encoded Mitochondrial Gene Duplicate, Is Essential for Male Fertility in <i>Drosophila melanogaster</i> |
| title_full | <i>COX4-like</i>, a Nuclear-Encoded Mitochondrial Gene Duplicate, Is Essential for Male Fertility in <i>Drosophila melanogaster</i> |
| title_fullStr | <i>COX4-like</i>, a Nuclear-Encoded Mitochondrial Gene Duplicate, Is Essential for Male Fertility in <i>Drosophila melanogaster</i> |
| title_full_unstemmed | <i>COX4-like</i>, a Nuclear-Encoded Mitochondrial Gene Duplicate, Is Essential for Male Fertility in <i>Drosophila melanogaster</i> |
| title_short | <i>COX4-like</i>, a Nuclear-Encoded Mitochondrial Gene Duplicate, Is Essential for Male Fertility in <i>Drosophila melanogaster</i> |
| title_sort | i cox4 like i a nuclear encoded mitochondrial gene duplicate is essential for male fertility in i drosophila melanogaster i |
| topic | nuclear-encoded mitochondrial gene gene duplication CRISPR knockout <i>COX4L</i> spermatogenesis <i>Drosophila melanogaster</i> |
| url | https://www.mdpi.com/2073-4425/13/3/424 |
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