A human mitochondrial transcription factor is related to RNA adenine methyltransferases and binds S-adenosylmethionine

Abstract

A critical step toward understanding mitochondrial genetics and its impact on human disease is to identify and characterize the full complement of nucleus-encoded factors required for mitochondrial gene expression and mitochondrial DNA (mtDNA) replication. Two factors required for transcription initiation from a human mitochondrial promoter are h-mtRNA polymerase and the DNA binding transcription factor, h-mtTFA. However, based on studies in model systems, the existence of a second human mitochondrial transcription factor has been postulated. Here we report the isolation of a cDNA encoding h-mtTFB, the human homolog of Saccharomyces cerevisiae mitochondrial transcription factor B (sc-mtTFB) and the first metazoan member of this class of transcription factors to which a gene has been assigned. Recombinant h-mtTFB is capable of binding mtDNA in a non-sequence-specific fashion and activates transcription from the human mitochondrial light-strand promoter in the presence of h-mtTFA in vitro. Remarkably, h-mtTFB and its fungal homologs are related in primary sequence to a superfamily of N6 adenine RNA methyltransferases. This observation, coupled with the ability of recombinant h-mtTFB to bind S-adenosylmethionine in vitro, suggests that a structural, and perhaps functional, relationship exists between this class of transcription factors and this family of RNA modification enzymes and that h-mtTFB may perform dual functions during mitochondrial gene expression.

FIG. 1.

Alignment of the human CGI-75 protein with sc-mtTFB and a putative S. pombe sc-mtTFB homolog. Shown is a ClustalW (40) alignment of the amino acid sequence of sc-mtTFB (S. cerevisiae) with putative human (Homo sapiens) and fission yeast (S. pombe) homologs identified in Blastp searches. Amino acids highlighted in gray are identical or chemically similar in all three species. Pairwise comparisons are denoted as follows. Asterisks below the H. sapiens or S. cerevisiae sequence indicate identical amino acid identity between the H. sapiens and S. pombe or S. cerevisiae and S. pombe proteins, respectively. Carets denote chemically similar amino acids in the same manner designated according to the following matrix: S/T, R/K, D/E, N/Q, F/Y/W, I/V/L/M.

FIG. 2.

Human CGI-75 is a mitochondrial protein. The mitochondrial localization of a CGI-75::EGFP fusion protein is shown. HeLa Tet-On cells were transiently transfected with a plasmid (pTRE2-HBGFP) that expresses a CGI-75::EGFP fusion protein and stained with the mitochondrion-specific dye Mitotracker Red. Shown are two representative transfected cells analyzed by fluorescence microscopy for EGFP fluorescence in green (labeled GFP), Mitotracker fluorescence in red (labeled MT), and a merge of the two signals (labeled merge), in which a yellow color indicates colocalization. In the panels on the right, the asterisks indicate the signals derived from a cell that was not transfected with the plasmid. As expected, this cell exhibited Mitotracker fluorescence but not EGFP fluorescence.

FIG. 3.

Putative nuclear transcription factor binding sites in the genomic region immediately upstream of the CGI-75 gene. Transcription factor binding sites identified using the TFSEARCH algorithm to access the TRANSFAC database (45) are indicated as follows: SP-1, NFκ-B, and AP-1 are underlined; MZF-1 is printed in bold type; and NRF-2 is highlighted in gray. The sequence shown is the CGI-75 sense DNA strand, and the first two codons of the CGI-75 gene are indicated by lowercase italics.

FIG. 4.

Recombinant human CGI-75 protein binds DNA in a non-sequence-specific manner. An autoradiogram from a gel mobility shift assay (see Materials and Methods) using recombinant GST-purified CGI-75 protein and an mtDNA probe containing the mitochondrial LSP is shown. Plus and minus signs indicate the presence and absence of CGI-75 protein and the nonspecific competitive inhibitor poly(dI-dC) in the sample, respectively. Location of the unbound DNA probe is indicated by the arrow, and the position of the retarded CGI-75-bound probe is indicated by the arrowhead. Thirty nanograms of poly(dI-dC) was added where indicated, and the amount of CGI-75 in each reaction is indicated at the top of the figure (numbers indicate nanograms of CGI-75 added).

FIG. 5.

Recombinant human CGI-75 protein activates transcription from the human mitochondrial LSP. Mitochondrial transcription was assayed using an in vitro runoff transcription assay that measures specific transcription initiation from the human LSP. The resulting product is a discrete radio-labeled RNA transcript (indicated by an arrowhead) that begins at the LSP and extends to the end of the linear DNA template (10). Protein components present (+) or absent (−) in each reaction are indicated at the top of the figure. All reactions contained 5 μl of an mtTFA-dependent mitochondrial extract containing human mtRNA polymerase (HeLa mt extract). Where indicated, 2 μl of gel-purified and renatured h-mtTFA was added to the reaction mixture. Reaction mixtures contained recombinant human CGI-75 protein in the following amounts: lane 2, 20 ng; lane 4, 20 ng; lane 5, 16 ng; lane 6, 12 ng. The fact that decreasing amounts of CGI-75 were added in the last three reaction mixtures (lanes 4 to 6) is indicated at the bottom of the figure by the decreasing slope of the triangle from left to right.

FIG. 6.

Human mtTFB (CGI-75) is closely related to RNA adenine methyltransferases. An alignment of the h-mtTFB amino acid sequence with RNA adenine methyltransferase enzymes from M. loti (labeled M. loti t'ferase) and E. coli (E. coli Ksg1) is shown. Amino acids highlighted in gray boxes are identical or chemically similar in all three proteins according to the matrix designated in the legend to Fig. 1. Groups of residues that are boxed are sequence motifs (motifs I to VIII) that are implicated as important structural or catalytic features of RNA methyltransferases (1). Asterisks indicate amino acids that are conserved between sc-mtTFB, h-mtTFB, and the putative S. pombe mtTFB as denoted in Fig. 1.

FIG. 7.

Recombinant h-mtTFB (CGI-75) binds S-adenosylmethionine. The results of a solid-phase S-adenosylmethionine-binding assay are shown. The amount (in counts per minute) of ³H-SAM bound to beads containing either a GST peptide (GST) or the GST::CGI-75 fusion protein (GST-CGI75) was determined in three experimental trials. The average of these three experiments is plotted on the ordinate, with ± one standard deviation from the mean indicated by brackets.