Mitochondrial cysteinyl-tRNA synthetase is expressed via alternative transcriptional initiation regulated by energy metabolism in yeast cells

Abstract

Eukaryotes typically utilize two distinct aminoacyl-tRNA synthetase isoforms, one for cytosolic and one for mitochondrial protein synthesis. However, the genome of budding yeast (Saccharomyces cerevisiae) contains only one cysteinyl-tRNA synthetase gene (YNL247W, also known as CRS1). In this study, we report that CRS1 encodes both cytosolic and mitochondrial isoforms. The 5 complementary DNA end method and GFP reporter gene analyses indicated that yeast CRS1 expression yields two classes of mRNAs through alternative transcription starts: a long mRNA containing a mitochondrial targeting sequence and a short mRNA lacking this targeting sequence. We found that the mitochondrial Crs1 is the product of translation from the first initiation AUG codon on the long mRNA, whereas the cytosolic Crs1 is produced from the second in-frame AUG codon on the short mRNA. Genetic analysis and a ChIP assay revealed that the transcription factor heme activator protein (Hap) complex, which is involved in mitochondrial biogenesis, determines the transcription start sites of the CRS1 gene. We also noted that Hap complex-dependent initiation is regulated according to the needs of mitochondrial energy production. The results of our study indicate energy-dependent initiation of alternative transcription of CRS1 that results in production of two Crs1 isoforms, a finding that suggests Crs1's potential involvement in mitochondrial energy metabolism in yeast. Aminoacyl-tRNA synthetase (ARS) 2 is an ancestral housekeeping enzyme found in all organisms. ARSs are essential for This work was supported in part by Grants-in-Aid for Scientific Research (S), (B), and (C) and for Young Scientists (B) 18H04794 and 18H02621 (to H. M.), 15K21759 and 18H05277 (to T. A.), 17K08619 (to T. I.), and 17K15408 (to A. N.) and Grant-in-Aid for Scientific Research on Innovative Areas 26111008 (to T. A.) from the Ministry of Education, Sciences, Sports, and Technology (MEXT) Japan and by AMED under Grants JP19gm5010002 (to H. M.) and JP18fm0208029 (to T. A.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S4, Tables S1 and S2, and experimental procedures.

Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP

“…This definitely casts in doubt the previous proposal of the existence of a cQRS mitochondrial echoform ( Rinehart et al, 2005 ). In agreement with our results ( Figure 3C ), mitochondrial echoforms of cCRS were also detected in a recent study and shown to result from alternative transcription and translation starts ( Nishimura et al, 2019 ), thereby unraveling how mtCRS is expressed from the CRS1 gene and rationalizing how mitochondrial Cys-tRNA Cys is produced.…”

“…Interestingly, we discovered that two of them, cytosolic phenylalanyl-tRNA synthetase 2 (cFRS2) and cytosolic histidinyl-tRNA synthetase have a dual localization. We also confirmed the recently reported dual cellular location of cytosolic cysteinyl-tRNA synthetase (cCRS) ( Nishimura et al, 2019 ). We further demonstrate that our yeast BiG Mito-Split-GFP strain can be used to better define non-conventional mitochondrial targeting sequences and to probe the mitochondrial importability of proteins from other eukaryotic species (human, mouse and plants).…”

“…Since the existence of a fully functional mtFRS has been demonstrated ( Koerner et al, 1987 ), it is possible that supernumerary mte cFRS2 we identified is not necessary for charging mitochondrial tRNA Phe but exerts some non-canonical functions, in addition to its role in cytosolic protein synthesis. The mitochondrial fluorescence triggered by expression of N 100 cCRS β11ch suggests that this part of cCRS harbors a MTS, which has recently been proposed ( Nishimura et al, 2019 , see Discussion). The mitochondrial fluorescence triggered by cyte cHRS β11ch is more intriguing.…”

Characterization of Polysulfides, Polysulfanes, and Other Unique Species in the Reaction between GSNO and H2S

“…Kimura found that 3-mercaptopyruvate sulfurtransferase and cysteine aminotransferase localized to a vascular endothelium produce H 2 S and persulfides H 2 S n (n = 1–3) [12], as well as cysteine- and glutathione-persulfides [13]. Akaike et al identified cysteinyl-tRNA synthetases that generate cysteine polysulfides (CysSS n H) which are integrated into proteins during translation [14,15]. These polysulfides were shown to be superior nucleophiles and reductants, and capable of regulating electrophilic cell signaling mediated by 8-nitroguanosine 3′,5′-cyclic monophosphate [16,17].…”

Localization and RNA Binding of Mitochondrial Aminoacyl tRNA Synthetases

“…Few aaRS genes, from various organisms, were found to utilize this route. Few examples were found in S. cerevisiae : CysRS gene generates two transcripts, and synthesis of the longer, MTS-containing transcript is coordinated with mitochondrial energy demands through binding of the heme regulatory transcription factor Hap1 [ 54 ]. ValRS gene produces two distinct mRNA products.…”