Divergent evolution of the paralogous human mitochondrial transcription factors, h-mtTFB1 and h-mtTFB2, to fulfill unique functions in mitochondrial gene expression, biogenesis, and retrograde signaling Open Access

Cotney, Justin Lee (2008)

Permanent URL: https://etd.library.emory.edu/concern/etds/n296wz42b?locale=en
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Abstract

Mitochondria are essential organelles, resulting from an ancient endosymbiosis that are found in virtually all eukaryotes. To maintain proper function, human mitochondria have maintained distinct genomes and a dedicated gene expression system. The human mitochondrial transcription system consists of three types of proteins: POLRMT, a bacteriophage-like RNA polymerase; h-mtTFA, a mitochondrial DNA binding protein; and the mtTFBs, two factors that provide a physical link between h-mtTFA and POLRMT required for transcription initiation. The human mtTFBs, h-mtTFB1 and h-mtTFB2, are unique transcription factors exhibiting homology with N6-adenine RNA dimethyltransferases. Phylogenetic analysis suggests a very early duplication of the endosymbiont KsgA gene as the source of these two paralogs. Consistent with this ancestry, I demonstrate that both h-mtTFB1 and h-mtTFB2 have maintained RNA methyltransferase activity. Overexpression of h-mtTFB1 in human cells increases the level of methylated 12S rRNA and induces mitochondrial mass. h-mtTFB2 overexpression elevates mitochondrial DNA levels, transcripts, mass, membrane potential, and surprisingly induces a coordinate increase in h-mtTFB1 expression. This indicates that h-mtTFB1 is the major 12S methyltransferase and h-mtTFB2 is involved primarily in mitochondrial transcription. These results also suggest a major role for these factors in coordinating mitochondrial biogenesis. By combining their activities, a robust remodeling of the mitochondrial compartment occurs, increasing organelle mass and greater respiratory capacity. This response is dependent specifically upon methylation activity of h-mtTFB1 and indicates that the methylation status of 12S rRNA is a metric for mitochondrial function. In support of this idea cells harboring the mtDNA mutation A1555G, a mutation in 12S rRNA linked to non-syndromic deafness, have elevated methylated 12S rRNA and phenotypes similar to those associated with h-mtTFB1 overexpression. I propose that 12S rRNA methylation status is regularly monitored by the cell and either induces expression of h-mtTFB1 to improve overall methylation or induces mitochondrial mass in preparation for more OXPHOS complexes to be produced by fully methylated mitochondrial ribosomes. Altogether this research indicates that these factors are intimately involved in regulating mitochondrial biogenesis. Inappropriate modulation of their levels or activities might contribute to human disease by producing mitochondria deficient in transcription, translation, or respiration.

Table of Contents

Chapter I: Origins and evolution of mitochondria ........................................1
1.1 Origins of mitochondria..............................................................................3
1.2 Endosymbiosis ...........................................................................................8
1.3 Mitochondrial genomes ............................................................................10


Chapter II: Mitochondrial biology ....................................................................15
2.1 Mitochondria as metabolic crossroads .................................................16
2.1.1 Essential processes ..............................................................................16
2.1.2 OXPHOS ...................................................................................................18
2.2 Replication and inheritance of mitochondria.........................................23
2.2.1 Organelle...................................................................................................23
2.2.2 mtDNA........................................................................................................25
2.3 Signaling and apoptosis............................................................................27
2.4 Cooperation of two genomes ...................................................................30
2.5 Roles of mitochondria in human disease...............................................32


Chapter III: Mitochondrial gene expression...................................................35
3.1 Mammalian mitochondrial transcription machinery..............................39
3.1.1 POLRMT ....................................................................................................39
3.1.2 mtTFA .........................................................................................................41

3.1.3 mtTFB1 and mtTFB2 ...............................................................................44
3.1.3.1 Transcription factor activity ..................................................................44
3.1.3.2 Homology with rRNA methyltransferases.........................................46
3.1.3.3 Are the mtTFBs dual functional proteins in vivo? ............................48
3.2 Mitochondrial transcription .........................................................................51
3.2.1 Models of initiation....................................................................................52
3.2.2 Elongation and Termination ..................................................................54
3.3 Mitochondrial ribosomes and the h-mtTFB factors ..............................56


Chapter IV: Results............................................................................................59
4.1 Divergent evolution of the mitochondrial transcription factor B
family ...................................................................................................................62
4.1.1 Duplication of the ancestral rRNA
methyltransferase..............................................................................................62
4.1.2 Lineages of mtTFBs ...............................................................................62
4.1.3 Maintenance of rRNA methyltransferase activity................................69
4.2 Relative levels of the human mitochondrial transcription
machinery............................................................................................................76
4.2.1 Specifically identifying mtTFB1 and mtTFB2 via
immunoblotting..................................................................................................76
4.2.2 Estimation of levels of mitochondrial transcription
machinery. .........................................................................................................78
4.2.3 mtDNA copy number measurements.................................................81

4.3 Distinct biological roles of the mitochondrial transcription factor
B family................................................................................................................85
4.3.1 Over-expression of wild type mtTFB1 and mtTFB2
in HeLa cells......................................................................................................85
4.3.2 Processing and predicted cleavage site of mtTFB2 .......................85
4.3.3 Over-expression of mtTFB2 induces mtTFB1 levels ......................86
4.3.4 Effects of over-expression of mtTFB1 and mtTFB2
on mitochondrial transcripts, mtDNA levels, and
mitochondrial translation................................................................................91
4.3.5 Over-expression of mtTFB1 and mtTFB2 increases
sensitivity to kasugamycin..............................................................................94
4.3.6 Effects of over-expression of mtTFB1 and mtTFB2
on mitochondrial biogenesis and membrane
potential.............................................................................................................95
4.4 Methylation status of human mitochondrial 12S rRNA is a
component of a retrograde signaling pathway for mitochondrial
biogenesis. ................................................................................................... 101
4.4.1 Over-expression of methylation deficient mutant hmtTFB1
and h-mtTFB2................................................................................................ 101
4.4.2 Methyltransferase deficient h-mtTFB2 maintains
higher transcript levels and mtDNA copy number ................................. 105

4.4.3 Methylation activity of mtTFB1 is required for
stimulation of mitochondrial biogenesis and is a
component of a mitochondrial retrograde signal................................... 105


Chapter V: Discussion................................................................................. 113
5.1 Implications of divergent evolution and maintenance of dual
functions in the h-mtTFB proteins.............................................................. 114
5.2 Model of impact of mtTFB levels and activities on
mitochondrial transcription and translation ............................................ 118
5.3 Relative levels of mitochondrial transcription machinery and
mtDNA............................................................................................................. 122
5.4 Outcomes of altering mitochondrial transcription machinery
levels .............................................................................................................. 125
5.5 12S rRNA methylation is a component of a signaling pathway
for mitochondrial biogenesis..................................................................... 130
5.6 Closing remarks ................................................................................... 140


Appendix A: POLRMT and MRPL12........................................................... 141
Appendix B: mtDNA Levels and Effects on Abundance of Transcription
and Translation Machinery ......................................................................... 146
Appendix C: POLRMT and MYC.................................................................. 150
Appendix D: Materials and Methods.......................................................... 154
D.1 Plasmids................................................................................................. 154
D.2 Bacterial kasugamycin-resistance assays ..................................... 156
D.3 Primer extension analysis of bacterial 16S rRNA adenine
methylation..................................................................................................... 156
D.4 Phylogenetic analysis .......................................................................... 157
D.5 Purification of recombinant proteins.................................................. 157
D.6 HeLa cell growth and transfection ..................................................... 159
D.7 Antibody production and western analysis of whole-cell and
mitochondrial proteins ................................................................................ 160
D.8 Nucleic acid extraction, Northern blotting, Reverse transcriptase
Real-Time PCR, and mtDNA copy number analysis ............................ 162
D.9 Labeling of mitochondrial translation products in vivo................... 166
D.10 Kasugamycin sensitivity assays ..................................................... 167
D.11 FACS analysis...................................................................................... 167
D.12 Mitochondrial localization sequence and cleavage prediction.... 167
D.13 Primer extension analysis of human 12S rRNA ........................... 168
Appendix E: Sequence Alignments............................................................ 169
Appendix F: References .............................................................................. 186
Vita.................................................................................................................... 216


List of Tables
Table 1 Accession numbers of amino acid sequences used in this study......64
Table 2 Estimation of levels and ratios of the mitochondrial transcription
components in HeLa cells. ........................................................................................84


List of Figures
Figure 1 Human mitochondrial DNA.........................................................................14
Figure 2 Phylogenetic analysis of the B family of mitochondrial
transcription factors and rRNA adenine dimethyltransferases from
bacteria, archaea, and eukaryotes. ..........................................................................66
Figure 3 Phylogenetic analysis of the mtTFB family of mitochondrial
transcription factors. ..............................................................................................68
Figure 4 Functional complementation of E. coli KsgA rRNA
methyltransferase activity by h-mtTFB2, but not its S. cerevisiae
ortholog sc-mtTFB.................................................................................................72
Figure 5 SAM-dependent methylation of an evolutionarily conserved stemloop
in the E. coli 16S rRNA by h-mtTFB2, but not its S. cerevisiae
ortholog, sc-mtTFB................................................................................................75
Figure 6 mtTFB antibody specificity ..........................................................................77
Figure 7 Estimation of levels of the mitochondrial transcription machinery
in HeLa cells..........................................................................................................80
Figure 8 Measurement of absolute mtDNA copy number in HeLa cells. .....................83
Figure 9 h-mtTFB2 is processed in vivo and its over-expression in HeLa
cells results in a coordinated increase of h-mtTFB1, but not POLRMT or
h-mtTFA................................................................................................................89
Figure 10 Detection of elevated h-mtTFB1 transcript in h-mtTFB2 overexpressing
cells......................................................................................................90
Figure 11 Over-expression of h-mtTFB2 increases the steady-state levels of
mtDNA-encoded transcripts and proteins, and doubles mtDNA copy
number. .................................................................................................................93
Figure 12 Analysis of mitochondrial translation rates and kasugamycin
sensitivity in h-mtTFB1 and h-mtTFB2 over-expression HeLa cell lines................98
Figure 13 Analysis of mitochondrial biogenesis and membrane potential in
h-mtTFB1 and h-mtTFB2 over-expression HeLa cell lines. ................................. 100
Figure 14 Over-expression of methyltransferase deficient forms of hmtTFB1
and h-mtTFB2 ....................................................................................... 102
Figure 15 Methyltransferase activity is not required for transcript level
increases induced by h-mtTFB2 over-expression ................................................. 104
Figure 16 Over-expression of h-mtTFB1 leads to more methylated 12S
rRNA 108
Figure 17 Methyltransferase activity is critical for mitochondrial biogenesis
driven by h-mtTFB1 over-expression................................................................... 109
Figure 18 Both A1555G mtDNA mutant cybrids and h-mtTFB1 overexpressing
cell lines have elevated mitochondrial mass and decreased
total ROS............................................................................................................. 111
Figure 19 The ratio of methylated to unmethylated 12S rRNA is altered in
A1555G mtDNA mutant cybrids.......................................................................... 112
Figure 20 A putative mtDNA regulatory scheme based on h-mtTFB1 and hmtTFB2
having partially overlapping, but non-identical transcription and
methylation activities........................................................................................... 121
Figure 21 Mitochondrial biogenesis and membrane potential control by hmtTFB1
and h-mtTFB2 ....................................................................................... 139
Figure 22 Over-expression of MRPL12 in HeLa cells enhances the steady
state level of mtDNA-encoded transcripts. ........................................................... 145
Figure 23 Ethidium Bromide depletion of mtDNA reduces levels of
mitochondrial transcription machinery................................................................. 149
Figure 24 Regulation of POLRMT by MYC controls transcription from the
mitochondrial genome. ......................................................................................... 153
Supplemental Figure 1 Sequence alignment used to generate tree shown in
Figure 1. ............................................................................................................. 169
Supplemental Figure 2 Sequence alignment used to generate tree in Figure 2. ............. 180

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