Abstract
Recent studies on nuclear-encoded mitochondrial genes (N-mt genes) in Drosophila melanogaster 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., Cytochrome c oxidase 4-like (COX4L)—in order to understand its function and, consequently, the reason behind its retention in the D. melanogaster genome. The COX4L gene is a duplicate of the Cytochrome c oxidase 4 (COX4) gene of OXPHOS complex IV. While the parental COX4 gene has been found in all eukaryotes, including single-cell eukaryotes such as yeast, we show that COX4L is only present in the Brachycera suborder of Diptera; thus, both genes are present in all Drosophila species, but have significantly different patterns of expression: COX4 is highly expressed in all tissues, while COX4L has a testis-specific expression. To understand the function of this new gene, we first knocked down its expression in the D. melanogaster germline using two different RNAi lines driven by the bam-Gal4 driver; second, we created a knockout strain for this gene using CRISPR-Cas9 technology. Our results showed that knockdown and knockout lines of COX4L 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 COX4L-HA in the germline, but not when driving COX4-HA. In addition, ectopic expression of COX4L in the soma caused embryonic lethality, while overexpression in the germline led to a reduction in male fertility. COX4L-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 Drosophila sperm proteome, suggests that COX4L is a paralogous, specialized gene that is assembled in OXPHOS complex IV of male germline cells and/or sperm mitochondria.
Highlights
Introduction iationsMitochondria produce a large fraction of cellular energy, but are involved in a diverse set of cellular functions, such as metabolism [1], immune regulation [2] and cell death [3]
A phylogenetic tree using a maximum likelihood (ML) model (Figure 1) shows that the two genes cluster into two distinct clades, suggesting that they have been evolving separately since the origin of c oxidase 4-like (COX4L)
Unlike c oxidase 4 (COX4), COX4L is present in the Drosophila sperm proteome (DSP) [11], which suggests that this gene is important for sperm function, and may have a different function than its parental counterpart
Summary
Introduction iationsMitochondria produce a large fraction of cellular energy, but are involved in a diverse set of cellular functions, such as metabolism [1], immune regulation [2] and cell death [3]. Mitochondria are organelles in eukaryotic cells with their own DNA (mtDNA), within which, during its approximately 1.5 billion years of evolution, the mitochondrial genome has experienced many changes. Few genes have remained in the mitochondrial genome, which in most metazoans consists of 13 proteincoding genes, 2 rRNA genes, and 22 tRNA genes [4]. Because of this severe reduction in gene content, mitochondria import most of their proteins (N-mt proteins) from the cytoplasm. The N-mt genes encompass genes that had mitochondrial functions
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