Elucidation of separate, but collaborative functions of the rRNA methyltransferase-related human mitochondrial transcription factors B1 and B2 in mitochondrial biogenesis reveals new insight into maternally inherited deafness

Abstract

Mitochondrial biogenesis is controlled by signaling networks that relay information to and from the organelles. However, key mitochondrial factors that mediate such pathways and how they contribute to human disease are not understood fully. Here we demonstrate that the rRNA methyltransferase-related human mitochondrial transcription factors B1 and B2 are key downstream effectors of mitochondrial biogenesis that perform unique, yet cooperative functions. The primary function of h-mtTFB2 is mtDNA transcription and maintenance, which is independent of its rRNA methyltransferase activity, while that of h-mtTFB1 is mitochondrial 12S rRNA methylation needed for normal mitochondrial translation, metabolism and cell growth. Over-expression of h-mtTFB1 causes 12S rRNA hypermethylation, aberrant mitochondrial biogenesis and increased sorbitol-induced cell death. These phenotypes are recapitulated in cells harboring the pathogenic A1555G mtDNA mutation, implicating a deleterious rRNA methylation-dependent retrograde signal in maternally inherited deafness pathology and shedding significant insight into how h-mtTFB1 acts as a nuclear modifier of this disease.

Figure 1.

Ribosomal RNA methyltransferase activity is required for the functions of h-mtTFB1, but not h-mtTFB2 in HeLa cells. (A) Shown is a western blot of mitochondrial extracts from HeLa cells that over-express wild-type (h-mtTFB1 and h-mtTFB2 #8) and methyltransferase-deficient forms (h-mtTFB1 G65A #6 and h-mtTFB2 G105A #2) of h-mtTFB1 and h-mtTFB2. Blots were probed for h-mtTFB1 and h-mtTFB2, other components of the mitochondrial transcription machinery (h-mtTFA, POLRMT and MRPL12) and a mitochondrial loading control (HSP60) as indicated. A cell line transformed with an empty-vector (vector) served as the negative control line to which all others were compared. (B) Mitotracker Green fluorescence (a measure of mitochondrial mass) values in the cell lines described in (A). Shown is the mean fluorescence, normalized to that observed in the vector control cell line, which was given a value of 1.0. (C) Northern blots of total RNA from the cell lines in (A) using the mitochondrial gene probes indicated to the right. Ethidium bromide staining of the 28S cytoplasmic rRNA (at the bottom) indicates similar loading in the lanes. (D) Results of methylation-specific primer extension analysis of mitochondrial 12S rRNA from the cell lines in (A). A primer anti-sense to the 3′-end of the 12S rRNA (diagrammed to the right) was end-labeled and incubated with total mitochondrial RNA. Extension from this primer by AMV reverse transcriptase is blocked at dimethylated adenines, resulting in shorter products (solid triangles). If no methylation is present, extension reads through the stem-loop region and produces longer products (indicated at the top). The position of the unextended primer is indicated by an open triangle. The ratio of truncated (methylated)/extended (read through) products indicates of the relative methylation status of 12S rRNA. In this and all remaining figures t-tests were performed where appropriate. The error bars indicate ± 1 SD from the mean, and statistical significance is indicated as follows: *P < 0.05, **P < 0.005 and ***P < 0.0005.

Figure 2.

Increased mitochondrial mass, translation and activity in h-mtTFB2 over-expressing cells requires the coordinate up-regulation of h-mtTFB1. (A) Immunoblot analysis of mitochondrial extracts from HeLa cell lines over-expressing h-mtTFB2, with (+) or without (−) stable h-mtTFB1 knock-down by shRNA. Empty-vector transformed cells (vector) are also shown as negative controls. The blots were probed for h-mtTFB1, h-mtTFB2 and HSP60 (as a mitochondrial loading control). (B) Immunoblot analysis of whole-cell extracts from the cell lines in (A). Blots were probed for COXI (a representative mtDNA-encoded protein), VDAC (a nucleus-encoded mitochondrial protein that can be taken as one indicator of mitochondrial mass), and tubulin (loading control). The graph on the right shows mean Mitotracker Green fluorescence values from the same cell lines as a separate measure of mitochondrial mass. (C) Mitochondrial translation profiles of the cell lines described in (A). A representative autoradiogram of the indicated radiolabeled mtDNA-encoded translation products is shown. (D) Mitochondrial oxygen consumption and total cellular ATP in the cell lines described in (A). Both graphs represent measurements that were normalized to the values obtained in the empty-vector control cell lines, which were given a value of 1.0.

Figure 3.

The increase in mtDNA and mitochondrial transcripts caused by h-mtTFB2 over-expression does not require simultaneous up-regulation of h-mtTFB1. (A) Northern analysis of HeLa cell lines over-expressing h-mtTFB2, with (+) or without (−) stable h-mtTFB1 knock-down by shRNA. Empty-vector transformed cells (vector) are also shown as negative controls. The blots were probed for the mtDNA transcripts indicated on the right and nuclear 28S is shown as a loading control. (B) Real-time PCR analysis of mtDNA copy number from the same cell lines as in (A).

Figure 4.

h-mtTFB1 is required for optimal mitochondrial translation and activity, 12S rRNA accumulation, and normal cell growth. (A) Western blot of mitochondrial extracts from empty-vector transformed HeLa cell lines, with (+) or without (−) h-mtTFB1 knock-down by shRNA. Blots were probed for h-mtTFB1, h-mtTFB2 and HSP60 (a mitochondrial loading control). (B) Growth curves of the cell lines described in (A), plotted as the number of viable cells as a function of time in hours. (C) Mitochondrial translation profiles of the cell lines described in (A). (D) Mitochondrial oxygen consumption and total cellular ATP in the cell lines described in (A). (E) Northern blot of total RNA from the cell lines in (A).

Figure 5.

Cells carrying the deafness-associated A1555G mtDNA mutation, like those that over-express h-mtTFB1, exhibit mitochondrial 12S hypermethylation, aberrant mitochondrial biogenesis, and increased susceptibility to cell death. (A) Shown are the results of methylation-sensitive 12S rRNA primer extension reaction from total RNA of wild-type and A1555G mutant cybrids, exactly as described in the legend to Figure 1, except in this case read-through products (*) were terminating due the presence of ddGTP (to yield a product of known length for quantification purposes, see Materials and Methods). The right panel shows quantification of results with means and standard deviations calculated from six separate measurements. (B) Immunoblots of whole cell extracts from wild-type and A1555G mtDNA mutant cybrids were serially probed for mitochondrial h-mtTFB1, h-mtTFB2, COX1, COXII and VDAC and tubulin (as a loading control) as indicated. (C) Mitotracker Green fluorescence values from three separate samples from wild-type and A1555G cybrids. (D) Results of sorbitol-induced cell death assays of wild-type and A1555G cybrids (upper graph) and h-mtTFB1-overexpression and empty-vector (vector) control cell lines. Graphed are the mean fold changes in percent dying cells due to sorbitol treatment of the indicated cell lines.

Figure 6.

Models depicting the independent, yet coordinated roles of h-mtTFB1 and h-mtTFB2 in mitochondrial biogenesis and the proposed involvement of aberrant mitochondrial biogenesis due to 12S rRNA hypermethylation in the deafness-associated pathology of the A1555G mtDNA mutation. Based on the results of this study and published information, we propose (A) that h-mtTFB1 and h-mtTFB2 are key regulators of mitochondrial biogenesis, each with distinct, but normally coordinated functions (h-mtTFB1: 12S methylation, translation and mass regulation; h-mtTFB2: transcription and transcription-primed DNA replication). Thus, h-mtTFB1 and h-mtTFB2 are likely key downstream mediators of characterized mitochondrial biogenesis signaling pathways (e.g. those mediated by PGC-1 family of regulators, cyclin D1 and E2F1). As h-mtTFB1 and h-mtTFB2 are known targets of nuclear-respiratory factors (NRF-1 and NRF-2), signaling can go through these factors. However, we acknowledge the possibility of other mediators (?) or direct effects from these factors (arrow bypassing intermediaries), and likely also other biogenesis pathways, such as those mediated by the PGC family and NRFs (left). The arrow between h-mtTFB2 and h-mtTFB1 represents the retrograde signal we have identified that allows up-regulation of h-mtTFB1 in response to increased h-mtTFB2. Thus, in principle, both factors could be activated by pathways that initially only stimulate h-mtTFB2. In (B) we postulate that the A1555G mtDNA mutation results in hypermethylation the mitochondrial 12S rRNA (most likely due to change in the conformation of the substrate) that elicits a retrograde signal that increases mitochondrial mass without coordinately up-regulating mitochondrial gene expression and mtDNA replication (i.e. unscheduled/aberrant mitochondrial biogenesis). This, in turn, results in heightened susceptibility to stress-induced hair cell pathology and cell death that contributes to the increased incidence in irreversible deafness in patients with the A1555G mutation.