Effect of Exercise Intensity on Isoform-Specific Expressions of NT-PGC-1α mRNA in Mouse Skeletal Muscle
Figure 1
Expression and identification of NT-PGC-1α-a, NT-PGC-1α-b, and NT-PGC-1α-c and PGC-1α-a, PGC-1α-b, and PGC-1α-c isoforms. (a) Schematic structure of the 5′-region of the murine PGC-1α gene. The schematic structure was slightly adapted from Miura et al. [9]. Boxes indicate promoters, exons, and lines introns. The coding regions and untranslated regions are shown in gray and white, respectively. The proximal promoter (PProx.) drives the transcription from exon 1a to produce NT-PGC-1α-a mRNA and PGC-1α-a mRNA. The alternative promoter (PAlt.) drives the transcriptions from alternative exon 1b to generate NT-PGC-1α-b and PGC-1α-c mRNA and PGC-1α-b and PGC-1α-c mRNA. (b) Three different isoforms of NT-PGC-1α (NT-PGC-1α-a, NT-PGC-1α-b, and NT-PGC-1α-c) are produced by different exon usage and alternative 3′ splicing at the intron between exons 6 and 7a which contains an in-frame stop codon TAA (red). Three different isoforms of PGC-1α (PGC-1α-a, PGC-1α-b, and PGC-1α-c) are produced by different exon usage and normal 3′ splicing at the intron between exons 6 and 7b. (c) The schematic diagram of amino acid sequences of NT-PGC-1α-a, NT-PGC-1α-b, and NT-PGC-1α-c and PGC-1α-a, PGC-1α-b, and PGC-1α-c. The difference in amino acid sequences among NT-PGC-1α-a, NT-PGC-1α-b, and NT-PGC-1α-c is only in N-terminus shown in gray color, and the remaining sequences of NT-PGC-1α-a, NT-PGC-1α-b, and NT-PGC-1α-c are identical to each other. (d) Schematic diagrams of primer design for detection of NT-PGC-1α-a, NT-PGC-1α-b, and NT-PGC-1α-c mRNA and PGC-1α-a, PGC-1α-b, and PGC-1α-c mRNA. Primer pairs are depicted to the left, exons 1–7 of PGC-1α gene are depicted to the middle, and the corresponding names of NT-PGC-1α and PGC-1α splice variants are stated to the right. Forward primers located in exon 1a (ex 1a) for NT-PGC-1α-a and PGC-1α-a, exon 1b (ex 1b) for NT-PGC-1α-b and PGC-1α-b, and exon 1b′-exon 2 (ex 1b′/ex 2) for NT-PGC-1α-c and PGC-1α-c were combined with reverse primers for NT-PGC-1α (ex 7a) and PGC-1α (ex 6/ex 7b). Reverse primers for total NT-PGC-1α (ex 7a) and total PGC-1α (ex 6/ex 7b) were combined with a common forward primer located in the junction of exons 5 and 6 (ex 5/ex 6). The detailed sequences of all primers above mentioned are listed in Table 1. (e) The slopes of the resulting standard curves relating log mass of NT-PGC-1α-a, NT-PGC-1α-b, NT-PGC-1α-c, PGC-1α-a, PGC-1α-b, and PGC-1α-c cDNA to cycle threshold are −3.968, −3.787, −3.748, −3.619, −3.837, and 3.868. (f) RT-PCR products of NT-PGC-1α-a, NT-PGC-1α-b, and NT-PGC-1α-c mRNA and PGC-1α-a, PGC-1α-b, and PGC-1α-c mRNA on agarose gel. The bands correspond to the calculated amplicon sizes: PGC-1α-a (770 bp), PGC-1α-b (800 bp), PGC-1α-c (768 bp), NT-PGC-1α-a (796 bp), NT-PGC-1α-b (831 bp), and NT-PGC-1α-c (799 bp). The normal PCR reaction conditions were 95°C for 10 min, 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min, and then 72°C for 5 min.
