5. Results
5.2 Functional studies on MTERFD1 and MTERFD3 (III)
5.2.1 MTERFD1 and MTERFD3 are mitochondrial proteins (III)
For MTERFD1 and MTERFD3 studies I established inducible epitope-tagged fusion protein expressing cell lines using the Flp-In™ T-Rex™ -293 cell system (HEK293T cells were also transiently transfected with the MTERFD3-FLAG expression construct). The expression of the epitope-tagged variants of MTERFD1 and MTERFD3 was verified by means of Western blotting. The size of the MTERFD3-HA or MTERFD3-FLAG protein was as predicted, roughly 43 kDa, when the epitope tag size has been taken into account and the mitochondrial targeting sequence has been removed. MTERFD1-FLAG was observed to migrate slightly slower than expected, approximately 47 kDa (Figure 5.15A).
The intracellular localization of the fusion proteins was studied by means of immunocytochemistry of transiently transfected cells. Both FLAG epitope-tagged proteins were mitochondrially targeted in vivo based on colocalization with Mitotracker Red. MTERFD1-FLAG but not MTERFD3-FLAG showed a distinctive punctuate staining pattern that suggested it is localized in nucleoids (Figure 5.15B).
Figure 5.15. Epitope tagged MTERFD1 and MTERFD3 are efficiently over-expressed in human cells and mitochondrially targeted. A) Flp-In™ T-Rex™ -293 cells were doxycycline induced for 48 h to express either epitope tagged MTERFD1-FLAG or MTERFD3-HA. HEK293T cells were transiently transfected (tr) with MTERFD3-FLAG and compared to mock transfected.
Western blots using total protein extracts were probed with anti-FLAG or anti-HA antibodies. B) HEK293T cells were transiently transfected with MTERFD1-FLAG or MTERFD3-FLAG for immunocytochemistry and 24 h after transfection probed with anti-FLAG antibody and counterstained with Mitotracker Red. Merge figure (created using false colours) shows that the stains colocalise. Reprinted from original article (III), copyright (2010), by permission of Springer.
5.2.2 MTERFD1 and MTERFD3 do not bind mtDNA sequence specifically (III)
SELEX and EMSA were used to study whether MTERFD1 and MTERFD3 bind DNA in sequence-specific manner. Previously I used SELEX successfully to look for the
preferred binding motif for MTERF-MycHis. The same conditions were used to look for binding motifs for MTERFD1-FLAG and MTERFD3-HA. However, SELEX results were negative in both cases after 7 rounds of selection. Two independent experiments were carried out for both proteins. 69 and 15 sequences were obtained for MTERFD1-FLAG whereas 25 and 29 clones were sequenced for MTERFD3-HA, and these sequences did not differ from the negative controls (35 clones sequenced).
A series of EMSA assays was carried out using mitochondrial extracts from cells transiently transfected with either MTERFD1 or MTERFD3 or from induced MTERFD1-FLAG or MTERFD3-HA Flp-In™ T-Rex™ -293 cells and using the same probes as was used for MTERF-MycHis EMSAs. The whole non-coding region (NCR) was checked for MTERFD1-FLAG and MTERFD3-HA binding as well as the canonical MTERF binding site at the 16S/tRNALeu(UUR)/ND1 gene junction region, the IQM tRNA cluster together with the C-terminal portion of ND1, OL and the ATPase6/COIII, ND5/ND6 and tRNAPro/tRNAThr gene junction sites that are all fragments where MTERF and its homologues or other mtDNA binding proteins have previously been reported or suggested to bind. However, there was no sign of mtDNA binding activity for either of the proteins studied, since no novel protein-DNA complexes were observed nor any clear changes in mobility were visible. Also the effect of over-expressing MTERFD1-FLAG or MTERFD3-HA on MTERF-MycHis binding was studied but no novel or enhanced bands compared to control cells were observed. When supershift assays are considered there were no any changes in mobility of the complexes in MTERFD1-FLAG over-expressing cells whereas cells over-expressing MTERFD3-HA did yield a supershift with anti-HA antibody when probed with 4 fragments of the promoter region or with ATPase6/COIII gene junction site covering fragment. Thus, although neither EMSA nor SELEX revealed plausible binding sites for either MTERFD1 or MTERFD3, MTERFD3 may associate with DNA-binding complexes in some specific regions of the genome. My findings indicate therefore that these proteins do not directly bind mtDNA but might be able to do so by interacting with other proteins (Figure 5.16).
A B
Figure 5.16. MTERFD1 and MTERFD3 binding in mitochondrial DNAin vitro. A) 150 bp long probes used in B) as illustrated. Promoters are denoted as PL, PH1 and PH2. OH is the heavy strand replication origin and terminus; tRNA coding genes denoted with hatched bars, upper and lower bars for encoded genes on heavy and light strand, respectively, protein coding gene denoted with grey bar. B)-F) Series of EMSA gels using crude mitochondrial protein extracts from Flp-In™ T-Rex™ -293 cells transfected with MTERFD1-FLAG (D1-FLAG) or MTERFD3-HA (D3-HA) +/- doxycycline induced expression of protein of interest or MTERF-MycHis stably transfected cells (MTERF-MH) and EMSA probes as indicated. Supershift assay was carried out using the following antibodies: anti-FLAG (f), anti-HA (h) and anti-Myc (m). E) Complex formed by ATPase6/COIII oligonucleotide probe (denoted with an arrow) is modestly enhanced by MTERFD3-HA over-expression and partially supershifted by antibody. F) Arrow indicates the complex formed by MTERF-MycHis alone. Reprinted from original article (III), copyright (2010), by permission of Springer.
5.2.3 MTERFD1 and MTERFD3 downregulate mtDNA copynumber (III)
To investigate whether MTERFD1 and MTERFD3 have any role in mtDNA replication given the preceding results on MTERF, I tested whether altering the expression levels thereof affects the mtDNA copynumber. The effect of over-expressing MTERFD1-FLAG and MTERFD3-HA was studied over 7 days. MTERFD1-FLAG expressing cells showed progressive downregulation of the mtDNA copynumber which was statistically significant on day 7 (Figure 5.17A). On the other hand the induction of MTERFD3-HA
gave only a transient decrease of the mtDNA copynumber, at the boundaries of statistical significance (Figure 5.17B). When the proteins were knocked down in HEK293T cells there was no effect on mtDNA copynumber (Figure 5.17C).
Figure 5.17. Effect of modulating MTERFD1 and MTERFD3 levels on mitochondrial DNA copynumber. A) Relative mtDNA copynumbers measured by means of Q-PCR for Flp-In™ T-Rex™ -293 cells over-expressing MTERFD1-FLAG or B) MTERFD3-HA. Normalised against copynumber of the uninduced cells. C) Grey bars show HEK293T cells silenced either for MTERFD1 or MTERFD3, not treated or transfected with empty shRNA vector (v). Black bars show MTERF-silenced cells (using siRNAs) in comparison with mock transfected cells. Data was normalized against relevant untransfected or mock-transfected cells. Here statistical significance is marked with * (p < 0.01) and borderline significance is denoted with # (p = 0.05, t-test).
Reprinted from original article (III), copyright (2010), by permission of Springer.
5.2.4 MTERFD1 and MTERFD3 influence mtDNA replication intermediates (III)
To study the role of MTERFD1 and MTERFD3 proteins in mtDNA replication we used 2DNAGE to study the effect of over-expressing MTERFD1-FLAG or MTERFD3-HA in Flp-In™ T-REx™ -293 cells compared to controls. Over-expression of either MTERFD1-FLAG or MTERFD3-HA produced a number of changes that indicated that over-expression of either leads to impaired completion of replication. The over-over-expression of MTERFD3-HA had a stronger effect than that of MTERFD1-FLAG (Figure 5.18A).
For this assay mtDNA was digested withPvuII, which cuts mtDNA once, about 2.5 kb upstream of the replication terminus near OH. Schematic drawings of the replication intermediates are shown in Figure 5.18C. Multi-junctional molecules, interpreted as double Y intermediates, were observed to be upregulated, indicating that over-expression of either of the proteins has effect on the final steps of replication. When MTERFD3 was
over-expressed uncut or gapped circles (gc), that are released by the resolution step, were depleted. Lagging-strand DNA synthesis remains unfinished in the uncut circles at the PvuII restriction site. The bubble arc, represents an intermediate produced at an early stage of replication, was unchanged (Figure 5.18A).
The effect of over-expressing MTERFD1 and MTERFD3 in mtDNA replication intermediates was almost opposite to that of over-expressing MTERF. Over-expression of MTERF led (Figure 5.18A) to accumulation of bubble intermediates that are paused near the high-affinity MTERF binding site. Conversely both multi-junctional structures and gapped circles were depleted by MTERF over-expression. When either MTERFD1 or MTERFD3 was knocked down by means of RNAi, most of the mtRIs were preserved, but incomplete circles accumulated (Figure 5.18B).
Figure 5.18. The role of MTERFD1 and MTERFD3 in mitochondrial replication. A), B) 1 g of total nucleic acids, digested with PvuII and probed with (ND2 + ND4) probe, were analysed by 2DNAGE. Cells were harvested 48 h after induction with 10 ng/ml doxycycline or after transfection with shRNA constructs. Panels i and ii are reproductions of Figure 4D in I.
Replication intermediates are denoted with arrows and schematic drawings are shown in C), bubble arc (b), incomplete circles (ic), Y species (Y), broken replication intermediates (br), complex junctional forms (cj). Red arrows in B) indicate incomplete circles with extensive single-strandedness. C) Schematic drawings of the mitochondrial replication intermediates. Uncut PvuII site is marked with red cross. Reprinted from original article (III), copyright (2010), by permission of Springer.