Gene structure of human mitochondrial ATP synthase gamma-subunit. Tissue specificity produced by alternative RNA splicing.

C Matsuda,H Endo,S Ohta,Y Kagawa

doi:10.1016/s0021-9258(19)74556-6

C Matsuda, H Endo + Show 2 more

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https://doi.org/10.1016/s0021-9258(19)74556-6

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Abstract

We completely sequenced the human gene for ATP synthase gamma-subunit, which was approximately 23 kilobases long and was composed of 10 exons. Exons 1 and 2 encoded the N-terminal presequence required for mitochondrial import, while exons 9 and 10 encoded the C-terminal portions of mature protein. Enzymatic amplification of human heart and liver cDNAs using the polymerase chain reaction revealed two mRNA transcripts that were predicted to encode two 30-kDa isoforms of the gamma-subunit, which differed by the addition of a single amino acid (Asp273) at the C terminus of the liver type isoform. These two mRNA transcripts of the heart (H) type and liver (L) type were generated by alternative splicing of an exon. The same alternative splicing event was observed in bovine tissue. In human tissues, the H type mRNA devoid of exon 9 was expressed specifically in the heart and skeletal muscle, which require rapid energy supply. The L type mRNA was expressed in the brain, liver, kidney etc. Both transcripts were expressed in the skin, intestine, stomach, and aorta. This tissue specificity of transcript heterogeneity suggests the distinct functional or regulatory roles of the gamma-subunit isoforms in the catalysis of ATP synthase. This is the first report on tissue-specific isoforms generated by alternative splicing in an energy transducing mitochondrial protein.

Highlights

We completely sequenced the human gene for ATP structure have been described in bovine studies [10, 11].In synthase y-subunit, which was approximately 23 kil- humans, the cDNAs and the genomic genes that encode the obases long and was composed of 10 exons
Human adenine nucleotide translocators are encoded by three different genes, T1, T2, andT3, which are expressed ina tissue-specific manner [22,23,24,25]. cDNAs for tissue-specific isoforms have been detected in the mammalian proteolipid ( 2 6 ), a component of Fo complex, and in three mammalian subunits (VIa, VIIa, and VIII) of cytochrome c oxidase [27,28,29]
Probe Preparation and Genomic Library Screening-Genomic gene encoding the ATP synthase y-subunit was isolated from a human genomic library by a method of plaque hybridization using the cloned PCR’ product of the bovine y-subunit cDNA as the hybridization probe [11].To prepare the hybridization probe, sense and antisense strand oligonucleotides (5’-GCCGCAATGGATCCAAGTTCGA-3’, tion of an ATPase activity and for the in vitro assembly of 5’-CAGCTTGCTGAATTCTTCTTTAC-3’co;rresponding to nuthe F1 portion [6, 8,9]

Summary

RESULTS

Genomic Structure of the Human ATP Synthase y-Subunit Gene-To isolate thehuman gene that encoded the ATP synthase y-subunit,a human genomic library constructed in the vector XDASH I1 was screened using the bovine liver cDNA [11]synthesized by PCR as a hybridization probe. Blot analysis showed they contained the entire gene for the ATP synthase y-subunittogether with substantial 5'- and 3'flanking regions (Fig. 1). The location of exon sequences within the genomic clones was tentatively identified following mapping with two restriction enzymes, EcoRI and HindIII, and 32P-labeledfragments of the 5'- and 3'-portionsof the amplified bovine cDNA. Each fragment that had been digested with EcoRI and Hind111 was ATP Synthase ?-Subunit Gene and Tissue-specificSplicing. To identify the entire exon sequence, with the amplifiedhumancDNAfragments, the genomic the human ATP synthase y-subunit cDNA wasamplified clones of XHATPG5 and 21 were shown to contain the 5' end from aHeLa or HT29 cDNA library constructedin pcD2 and 3' endof the gene, respectively (Fig. 1). These amplified fragments were subcloned and the phage clones were subcloned and sequencedT.he complete sequenced (Fig. 2).

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DISCUSSION