Abstract
Mitochondrial transcription in the early Xenopus laevis embryo resumes several hours before active mtDNA replication, effectively decoupling mtDNA transcription and replication. This developmental feature makes Xenopus embryogenesis an appealing model system to investigate the regulation of mitochondrial transcription. Studies reported here refine our understanding of the timing, magnitude, and mechanism of this transcriptional induction event. Northern analyses of six mitochondrial mRNAs (normalized to mtDNA) reveal that transcript levels remain basal between fertilization and gastrulation and then undergo a coordinate induction, culminating in a 20-28-fold increase over egg levels by 48 h of development. Measurement of mitochondrial run-on transcription rates demonstrates a good correlation between transcription rates and transcript levels, showing that transcription itself is the primary determinant of transcript abundance. Experimental increases in mitochondrial ATP and energy charge also correlate with patterns of transcript levels and transcription rates, suggesting that developmental changes in the biochemical composition of the mitochondrial matrix could be regulating transcriptional activity. Consistent with this idea, transcriptional run-on rates in mitochondria of early embryos can be stimulated by the addition of tricarboxylic acid cycle intermediates to the run-on reaction. However, mitochondria of later stages do not show this response to the addition of metabolite. In combination, these data suggest that mitochondrial transcription is under metabolic regulation during early Xenopus embryogenesis.
Highlights
The results reveal that mitochondrial gene expression during early embryogenesis is regulated primarily by transcription
X. laevis provides an experimental window on this problem because mitochondrial transcription precedes resumption of mtDNA synthesis by at least 24 h
The timing, magnitude, and nature of this early transcriptional regulation were studied by measuring steady-state levels of six mRNAs and the two mitochondrial rRNAs between fertilization and 72 h of development
Summary
Plasmid Clones and Riboprobes—Gene-specific DNA templates for making hybridization probes and targets were constructed by cloning polymerase chain reaction-amplified fragments into the HincII site of pBS1 (ϩ/Ϫ) phagemid (Stratagene, La Jolla, CA). Isolation of Mitochondrial Nucleic Acids—For total RNA isolation, 100 eggs or embryos were thawed in 10 ml of lysis buffer (4 M guanidine thiocyanate, 100 mM sodium acetate, pH 5.0, 5 mM EDTA), incubated for 20 min at room temperature to facilitate dissociation of nucleoprotein complexes, extracted three or four times with phenol:chloroform: isoamyl alcohol (25:24:1, pH 7.0), loaded on 5.7 M CsCl cushions, and spun at 38,000 rpm for 24 h at 4 °C in an SW40 rotor to recover RNA pellets. To correct for differences in the yield of mitochondria obtained from the different developmental stages, 10% of the mitochondria in the run-on reactions were lysed, extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform, and processed for dot blotting (without ethanol precipitation) as described in under “RNA Analysis.”. Protein pellets obtained after trichloroacetic acid precipitation were resuspended in 10 mM Tris, pH 8.5, 0.5% SDS, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and the clarified supernatant was used for protein estimation [16]
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