A DNA replication-related element downstream from the initiation site of Drosophila selenophosphate synthetase 2 gene is essential for its transcription

Jin, Jing Shun; Baek, Seung-Hee; Lee, Hyesin; Oh, Moon Young; Koo, Yong Eui; Shim, Myoung Sup; Kwon, So Yeon; Jeon, Ik Soo; Park, So Young; Baek, Kwanghee; Yoo, Mi‐Ae; Hatfield, Dolph L.; Lee, Byeong Jae

doi:10.1093/nar/gkh569

Cited by 11 publications

(5 citation statements)

References 29 publications

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“…SL2 cell culture and DNA transfection of SL2 cells were performed as described previously [5,12] with minor modifications. Briefly, in a single transfection experiment, 2 µg of vector was mixed with 100 µl of dimethyldioctadecyl ammonium bromide (125 µg/ml) and 200 µl of HyQ SFX-Insect medium, incubated for 30 min at room temperature (20°C), and then added to a well of a six-well plate containing 6×10 6 cells and the cells were incubated for 48–72h at 25°C.…”

Section: Methodsmentioning

confidence: 99%

l(2)01810 is a novel type of glutamate transporter that is responsible for megamitochondrial formation

Shim

Kim

Lee

et al. 2011

Biochemical Journal

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l(2)01810 causes glutamine-dependent megamitochondrial formation when it is overexpressed in Drosophila cells. In the present study, we elucidated the function of l(2)01810 during megamitochondrial formation. The overexpression of l(2)01810 and the inhibition of glutamine synthesis showed that l(2)01810 is involved in the accumulation of glutamate. l(2)01810 was predicted to contain transmembrane domains and was found to be localized to the plasma membrane. By using 14C-labelled glutamate, l(2)01810 was confirmed to uptake glutamate into Drosophila cells with high affinity (Km =69.4 µM). Also, l(2)01810 uptakes glutamate in a Na+ -independent manner. Interestingly, however, this uptake was not inhibited by cystine, which is a competitive inhibitor of Na+ -independent glutamate transporters, but by aspartate. A signal peptide consisting of 34 amino acid residues targeting to endoplasmic reticulum was predicted at the N-terminus of l(2)01810 and this signal peptide is essential for the protein’s localization to the plasma membrane. In addition, l(2)01810 has a conserved functional domain of a vesicular-type glutamate transporter, and Arg146 in this domain was found to play a key role in glutamate transport and megamitochondrial formation. These results indicate that l(2)01810 is a novel type of glutamate transporter and that glutamate uptake is a rate-limiting step for megamitochondrial formation.

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Section: Methodsmentioning

confidence: 99%

l(2)01810 is a novel type of glutamate transporter that is responsible for megamitochondrial formation

Shim

Kim

Lee

et al. 2011

Biochemical Journal

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“…SL2 Cell Culture and RNA Interference-SL2 cell culture and preparation of double-stranded RNAs were carried out as described previously (43). For RNA interference, 0.25 ϫ 10 6 cells were plated on a 24-well plate in 0.5 ml of HyQ SFX-Insect media.…”

Section: Methodsmentioning

confidence: 99%

Elevation of Glutamine Level by Selenophosphate Synthetase 1 Knockdown Induces Megamitochondrial Formation in Drosophila Cells

Shim

Kim

Jung

et al. 2009

Journal of Biological Chemistry

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Although selenophosphate synthetase 1 (SPS1/SelD) is an essential gene in Drosophila, its function has not been determined. To elucidate its intracellular role, we targeted the removal of SPS1/SelD mRNA in Drosophila SL2 cells using RNA interference technology that led to the formation of vacuole-like globular structures. Surprisingly, these structures were identified as megamitochondria, and only depolarized mitochondria developed into megamitochondria. The mRNA levels of l(2)01810 and glutamine synthetase 1 (GS1) were increased by SPS1/SelD knockdown. Blocking the expression of GS1 and l(2)01810 completely inhibited the formation of megamitochondria induced by loss of SPS1/SelD activity and decreased the intracellular levels of glutamine to those of control cells suggesting that the elevated level of glutamine is responsible for megamitochondrial formation. Overexpression of GS1 and l(2)01810 had a synergistic effect on the induction of megamitochondrial formation and on the synthesis of glutamine suggesting that l(2)01810 is involved in glutamine synthesis presumably by activating GS1. Our results indicate that, in Drosophila, SPS1/SelD regulates the intracellular glutamine by inhibiting GS1 and l(2)01810 expression and that elevated levels of glutamine lead to a nutritional stress that provides a signal for megamitochondrial formation.Selenium is an essential trace element in the diet of humans and many other life forms. It provides many health benefits such as roles in preventing cancer and heart disease, serving as an antiviral agent, stimulating the immune system, reactive oxygen species (ROS) 4 scavenging, and male reproduction (1-6). Many of the benefits of selenium are most likely due to the presence of this element in selenoproteins as the amino acid selenocysteine (Sec) (7-9). Sec is the 21st amino acid in the genetic code (10 -12) and is incorporated into selenoproteins in response to UGA Sec codons (13,14). The active donor of selenium in Sec biosynthesis is monoselenophosphate (15), which is synthesized from selenite and ATP by an enzyme designated as selenophosphate synthetase (SPS) (16). There are two isoforms of SPS in higher eukaryotes, SPS1/SelD and SPS2, whereas only one type of SPS (SelD) exists in lower eukaryotes and eubacteria (17). The sequences of SPS1/SelD and SPS2 are highly conserved. For example, the amino acid sequence homology between human SPS1/SelD and SPS2 is 72% and that between Drosophila SPS1/SelD and SPS2 is ϳ45%. One of the major differences between SPS1/SelD and SPS2 is that SPS1/ SelD has an arginine at the position corresponding to Sec in SPS2 (18,19).Initially, both SPS1/SelD and SPS2 were thought to be involved in selenophosphate synthesis. However, it was subsequently shown that only SPS2 catalyzes selenophosphate synthesis. In in vitro experiments, SPS2 synthesized selenophosphate from selenide and ATP, but SPS1/SelD did not have this activity (20). Knockdown of SPS2 in NIH3T3 cells led to the loss of selenoprotein biosynthesis, whereas the inhibition of SPS1/ SelD...

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“…Humans express 25 selenoproteins, of which three are conserved in Drosophila (68)(69)(70)(71). Of the three Drosophila selenoproteins, selenophosphate synthetase 2 (SPS2) is broadly expressed and, intriguingly, is not only a selenoprotein but also performs a key role in selenocysteine biosynthesis by promoting the formation of the selenium donor, selenophosphate (72,73). Thus, loss of SPS2 impairs all selenoprotein synthesis.…”

Section: Alkbh8 Promotes Global and Selenoprotein Translationmentioning

confidence: 99%

tRNA modification enzyme-dependent redox homeostasis regulates synapse formation and memory

Madhwani,

Sayied,

Ogata

et al. 2023

Preprint

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Post-transcriptional modification of RNA regulates gene expression at multiple levels. ALKBH8 is a tRNA modifying enzyme that methylates wobble uridines in specific tRNAs to modulate translation. Through methylation of tRNA-selenocysteine, ALKBH8 promotes selenoprotein synthesis and regulates redox homeostasis. Pathogenic variants in ALKBH8 have been linked to intellectual disability disorders in the human population, but the role of ALKBH8 in the nervous system is unknown. Throughin vivostudies inDrosophila, we show that ALKBH8 controls oxidative stress in the brain to restrain synaptic growth and support learning and memory.ALKBH8null animals lack wobble uridine methylation and exhibit a global reduction in protein synthesis, including a specific decrease in selenoprotein levels. Loss ofALKBH8or independent disruption of selenoprotein synthesis results in ectopic synapse formation. Genetic expression of antioxidant enzymes fully suppresses synaptic overgrowth inALKBH8null animals, confirming oxidative stress as the underlying cause of dysregulation.ALKBH8animals also exhibit associative learning and memory impairments that are reversed by pharmacological antioxidant treatment. Together, these findings demonstrate the critical role of tRNA modification in redox homeostasis in the nervous system and reveal antioxidants as a potential therapy for ALKBH8-associated intellectual disability.Significance StatementtRNA modifying enzymes are emerging as important regulators of nervous system development and function due to their growing links to neurological disorders. Yet, their roles in the nervous system remain largely elusive. Throughin vivostudies inDrosophila, we link tRNA methyltransferase-regulated selenoprotein synthesis to synapse development and associative memory. These findings demonstrate the key role of tRNA modifiers in redox homeostasis during nervous system development and highlight the potential therapeutic benefit of antioxidant-based therapies for cognitive disorders linked to dysregulation of tRNA modification.

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A DNA replication-related element downstream from the initiation site of Drosophila selenophosphate synthetase 2 gene is essential for its transcription

Cited by 11 publications

References 29 publications

l(2)01810 is a novel type of glutamate transporter that is responsible for megamitochondrial formation

l(2)01810 is a novel type of glutamate transporter that is responsible for megamitochondrial formation

Elevation of Glutamine Level by Selenophosphate Synthetase 1 Knockdown Induces Megamitochondrial Formation in Drosophila Cells

tRNA modification enzyme-dependent redox homeostasis regulates synapse formation and memory

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