Abstract
In eukaryotes, the precise transcriptional and post-transcriptional regulations of gene expression are crucial for the developmental processes. More than 100 types of post-transcriptional RNA modifications have been identified in eukaryotes. The deposition of N6-methyladenosine (m6A) into mRNA is among the most common post-transcriptional RNA modifications known in eukaryotes. It has been reported that m6A RNA modification can regulate gene expression. The role of yeast m6A methyltransferase (Ime4) in meiosis and sporulation in diploid cells is very well proven, but its physiological role in haploid cells has remained unknown until recently. Previously, we have shown that Ime4 epitranscriptionally regulates triacylglycerol (TAG) metabolism and vacuolar morphology in haploid cells. Mitochondrial dysfunction leads to TAG accumulation as lipid droplets (LDs) in the cells; besides, LDs are physically connected to the mitochondria. As of now there are no reports on the role of Ime4 in mitochondrial biology. Here we report the important role played by Ime4 in the mitochondrial morphology and functions in Saccharomyces cerevisiae. The confocal microscopic analysis showed that IME4 gene deletion causes mitochondrial fragmentation; besides, the ime4Δ cells showed a significant decrease in cytochrome c oxidase and citrate synthase activities compared to the wild-type cells. IME4 gene deletion causes mitochondrial dysfunction, and it will be interesting to find out the target genes of Ime4 related to the mitochondrial biology. The determination of the role of Ime4 and its targets in mitochondrial biology could probably help in formulating potential cures for the mitochondria-linked rare genetic disorders.
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Introduction
In eukaryotes, the precise and controlled regulation of gene expression is crucial for the developmental processes. The post-transcriptional regulation that acts at the translational level is complementary to transcriptional regulation. The RNA-binding proteins play a key role in post-transcriptional regulation by controlling the mRNA levels (Jin and Neiman 2016; Berchowitz et al. 2013). Not only specific regulatory mechanisms but also cellular growth govern gene expression because both synthesis and degradation simultaneously determine mRNA levels (Chavez et al. 2016). The cellular growth depends on different metabolites. In eukaryotes, specific metabolites play key regulatory roles and thus directly connect metabolic status and cellular functions (Saint-Marc et al. 2015). S-Adenosylmethionine (SAM), an important metabolite, serves as the sole methyl donor for the methylation of histones, nucleic acids, and phospholipids (Ding et al. 2015). In addition, SAM is physiologically linked with lipid accumulation. In the previous study, we have shown that yeast m6A methyltransferases Ime4 epitranscriptionally regulates TAG metabolism and vacuolar morphology in haploid cells (Yadav and Rajasekharan 2017). Recently, studies have shown that mitochondrial dysfunction causes TAG accumulation and formation of LDs in the cells (Singh et al. 2016; Lee et al. 2013). The LDs play an important role in lipid metabolism and energy homeostasis through their interaction with mitochondria (Pu et al. 2011). The most obvious question that arises from these findings is, does TAG metabolism and mitochondria are physiologically linked via methylation? Therefore, in the present study, we studied the role of m6A methyltransferases Ime4 in the mitochondrial functions. Our data showed that IME4 gene deletion produces mitochondrial dysfunction. It will be interesting to find out the target genes of Ime4 which are directly involved in the mitochondrial biogenesis, morphology, and functions.
The m6A methyltransferases and their physiological significance
In eukaryotes, post-synthetic modifications of proteins, DNA, and RNA are common features. Unlike protein and DNA modifications, RNA modifications are not well studied (Schwartz et al. 2013). There are more than 100 types of post-synthetic RNA modifications known, yet our knowledge about their function and physiological significance is limited (Blanco and Frye 2014). The recent development of transcriptome-wide methods to identify RNA modifications such as 5-methylcytidine (m5C) and N6-methyladenosine (m6A) has created a new research field, the ‘epitranscriptome’. It became evident that these post-transcriptional RNA modifications regulate different fundamental cellular processes (Blanco and Frye 2014). The deposition of m6A into mRNA is among the most common post-transcriptional RNA modifications known in eukaryotes (Schwartz et al. 2013; Yue et al. 2015). N6-Adenosyl methyltransferases that introduce a tightly controlled deposition m6A into mRNA are found in almost all kingdoms of eukaryotic life (Yue et al. 2015; Dominissini et al. 2012). Like DNA methylation, it has been reported that m6A RNA modification can regulate gene expression (Fu et al. 2014; Zheng et al. 2013). Role of m6A RNA modification has also been suggested in other key functions such as RNA splicing (Jia et al. 2011), mRNA degradation (Harigaya et al. 2006), RNA stability (Zhang et al. 2010; Brennan and Steitz 2001), translational control (Tuck et al. 1999), meiosis (Schwartz et al. 2013), and cellular differentiation (Zhong et al. 2008).
The m6A methyltransferase in Saccharomyces cerevisiae
Since the m6A methyltransferases are fundamentally conserved throughout eukaryotes, S. cerevisiae is used as a model system to understand the biological relevance of m6A methylation. In S. cerevisiae, Ime4 is a counterpart of mammalian m6A methyltransferase (METTL3). The IME4 gene locus is transcribed into IME4 (sense RNA) and RME2 (the antisense RNA) transcripts depending on the cell type. The role of m6A methyltransferase (Ime4) in meiosis and sporulation in diploid cells is very well studied (Agarwala et al. 2012; Clancy et al. 2002), but its physiological role in haploid cells has remained unknown until recently. In the previous study (Yadav and Rajasekharan 2017), we have shown that Ime4 epitranscriptionally regulates triacylglycerol (TAG) metabolism and vacuolar morphology in haploid cells, independently of the MIS complex (RNA methylation complex consists of the Ime4, Mum2, and Slz1 proteins). The RNA modification-mediated regulation of genes is termed as the epitranscriptional regulation.
A few studies have demonstrated that yeast Ime4 has stationary phase-related functions when glucose is exhausted (Yadav and Rajasekharan 2017; Hongay et al. 2006). In yeast, TAG synthesis is a characteristic feature of the stationary growth phase (Horvath et al. 2011). Recently, studies have shown that defects in mitochondria cause TAG accumulation and formation of lipid droplets (LDs) in the cells (Singh et al. 2016; Lee et al. 2013). LDs are dynamic organelles and consist of a core of TAG and steryl esters surrounded by a phospholipids monolayer that has peripheral and embedded proteins (Farese and Walther 2009). LDs play an important role in the storage and mobilization of TAG. Several studies have shown that LDs are physically connected to the endoplasmic reticulum, mitochondria, and peroxisomes (Martin and Parton 2006; Goodman 2008; Murphy et al. 2009). The LDs play an important role in lipid metabolism and energy homeostasis through their physical connection with mitochondria (Pu et al. 2011). However, the physiological function of these contacts remains poorly understood. Studies of the interaction of LDs with mitochondria will provide new insights into the lipid metabolism and energy homeostasis as well as the pathophysiology of metabolic disorders. LDs have been seen attached to the mitochondria in adipocytes, hepatocytes, and skeletal muscle cells (Novikoff et al. 1980; Kalashnikova and Fadeeva 2006; Shaw et al. 2008). Recently, a study proposed that diacylglycerol (DAG), an intermediate of TAG metabolism, promotes mitochondrial fission (Frohman 2015). As it seems that TAG is metabolically linked with mitochondria and a recent study identified a physiological link between vacuole and mitochondria in yeast (Hughes and Gottschling 2012), we hypothesized a possible role of Ime4 in the mitochondrial functions.
The yeast m6A methyltransferase and mitochondrial functions
As glucose is being used up, the diauxic shift occurs, involving an increase in the expression of nuclear genes responsible for mitochondrial biogenesis (Ocampo et al. 2012). In yeast, fully developed and enlarged tubular mitochondria are characteristic features of stationary phase when glucose is exhausted (Aung-Htut et al. 2013). Therefore, in the present study, to understand the role of Ime4 in mitochondrial functions, the yeast cells were harvested from the stationary phase.
The yeast strains used in this work were procured from Euroscarf. The mitochondrial targeting pVT100U-mtGFP plasmid is the same as reported previously (Westermann and Neupert 2000). The growth and culture conditions were the same as reported previously (Yadav and Rajasekharan 2016). Briefly, a single colony of the yeast strains was precultured in 5 ml of YPD (yeast extract peptone dextrose) liquid medium (1% yeast extract, 2% peptone, 2% glucose, pH 6.5) in a 50-ml culture tube at 30 °C and was allowed to grow with constant shaking overnight. An equal quantity of yeast cells was taken from the preculture and subcultured in the required volume of synthetic minimal medium (SM, yeast nitrogen base: 6.7 g; amino acids drop-out mixture: 1.92 g; uracil: 76 mg/l; and 2% glucose; pH 6.5) at 30 °C. The yeast transformants harboring the pVT100U-mtGFP plasmids were precultured and subcultured in the SM − U + 2% glucose medium at 30 °C. The materials used in media preparation were purchased from HiMedia, Sigma-Aldrich, and Difco. The yeast transformation kit was obtained from Clontech.
The stationary-phase cells grown in SM were harvested to assess the mitochondrial morphology and functions. The cells from different genetic backgrounds overexpressing the pVT100U-mtGFP plasmids were collected, washed, and stained with the mitochondrial dye MitoTracker Orange CMTMRos (Life Technologies). Dye at the concentration of 100 nM in 10 mM HEPES buffer containing 5% glucose (pH 7.4) was used for the staining purpose. The images were captured with a Zeiss LSM 700 confocal laser scanning microscope. The microscopic imaging showed that IME4 gene deletion causes mitochondrial fragmentation (Fig. 1a). To assess the effect of IME4 gene deletion on mitochondrial functions, cytochrome c oxidase and citrate synthase activities were also measured. The kits for Yeast mitochondria isolation (MITOISO3), cytochrome c oxidase activity (CYTOCOX1), and citrate synthase activity (CS0720) were purchased from Sigma-Aldrich. The cytochrome c oxidase and citrate synthase activities were measured according to the manufacturer’s protocol. The ime4Δ strain showed a significant decrease in cytochrome c oxidase and citrate synthase activities compared to the wild-type cells (Fig. 1b, c).
Discussion and future perspectives
In the present study, we report that IME4 gene deletion causes mitochondrial dysfunction. Identification and determination of the target genes of Ime4 that are directly involved in the mitochondrial biogenesis, morphology, and functions will pave the way to understand the role of m6A methylation in mitochondrial biology. The mitochondria are vital organelles of the living cell. Essential metabolic reactions and the regulation of some signaling cascades take place in the mitochondria (Dimmer and Scorrano 2006). Defects in the mitochondrial morphology and functions and lipid metabolism have also been associated with the neurodegenerative disorders, such as Alzheimer’s disease, and Huntington’s disease (Lin and Beal 2006). The hereditary spastic paraplegia (HSP, also known as Strumpell-Lorrain disease), a heterogeneous group of genetic neurodegenerative disorders, has also been associated with the mitochondrial dysfunction. In humans, many mitochondrial-linked rare genetic diseases are associated with mutations in poorly characterized genes. Determination and characterization of these genes and their regulation are crucial for understanding and formulating cures for the rare genetic diseases. The determination of the role of Ime4 and its targets in mitochondrial morphology and functions probably could help in the development of therapeutic strategies focused on the m6A mRNA methylation.
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Acknowledgements
This work was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi, under the 12th five-year plan project LIPIC. P.K.Y. was supported by the student fellowship from CSIR, New Delhi. The corresponding author is a recipient of the JC Bose National Fellowship.
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RR conceived and initiated the project. RR and PKY designed the experiments. PKY executed the experiments and analyzed the data. PKY and RR discussed the data and wrote the paper.
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Communicated by M. Kupiec.
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Yadav, P.K., Rajasekharan, R. The m6A methyltransferase Ime4 and mitochondrial functions in yeast. Curr Genet 64, 353–357 (2018). https://doi.org/10.1007/s00294-017-0758-8
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DOI: https://doi.org/10.1007/s00294-017-0758-8