Transient overexpression of an alternative spliced isoform of mitochondrial transcription factor A (Δ5TFAM) affects mitochondrial transcription in the H1299 cell line
Mitochondrial transcription factor A (Tfam) is necessary for both transcription and maintenance of mitochondrial DNA, it is abundant enough to wrap the entire mtDNA and thus organizes a protein-DNA complex.
The gene is estimated to span about 10 kb in mouse, in human and in rat. The sequence of the human gene contains an open reading frame of 741 bp over seven exons and encodes a protein with a molecular weight of 25 kDa. A short mRNA of Tfam, lacking exon 5 (05Tfam), was found to be widely distributed in human tissues, representing 30% of the total Tfam transcript pool. Changes in the primary structure of the protein can alter the binding propertíes of proteins, influence their intracellular localization and modify their enzymatic activity and/or protein stability by diverse mechanisms as well as other characteristics (Stamm et al., 2005). 45Tfam isoform localization and its effect on mitochondrial transcription were studied.
To analyze the localization of 05Tfam isoform a chimeric protein consisting of A5Tfam and of the green fluorescent protein GFP as visualized tag has been expressed. In vitro expression was performed by using the TNT Quick Coupled Transcription/Translation System (Promega). Proteins obtained were observed by immunoblotting. To study the effects of the alternative spliced 05Tfam on the transcriptional activity, transient overexpression of either full length Tfam or 05Tfam in H1299 celi was carried out, and reactions of Sybr Green Reaf time-PCR were then performed to observe the behaviour of Tfam, 05Tfam and of the mitochondrial transcript COI.
The results reported show that mitochondrial transcription increases eìther after full length Tfam either after 05Tfam transfection although less efficiently for the second one. These results agree with the smaller capability of 05Tfam to bind mtDNA in comparison witthe smaller capability of 05Tfam to bind mtDNA in comparison with the full length isoform