Negative regulation of mitochondrial transcription by mitochondrial topoisomerase I

Stefan Sobek,Ilaria Dalla Rosa,Jürgen-Christoph Von Kleist-Retzow,Yves Pommier,Beatrice Bornholz,Frank Hillebrand,Faiza Kalfalah,Christian Mielke,Rudolf J Wiesner,Morten O Christensen,Heiner Schaal,Fritz Boege,Hongliang Zhang

doi:10.1093/nar/gkt768

Abstract

Mitochondrial topoisomerase I is a genetically distinct mitochondria-dedicated enzyme with a crucial but so far unknown role in the homeostasis of mitochondrial DNA metabolism. Here, we present data suggesting a negative regulatory function in mitochondrial transcription or transcript stability. Deficiency or depletion of mitochondrial topoisomerase I increased mitochondrial transcripts, whereas overexpression lowered mitochondrial transcripts, depleted respiratory complexes I, III and IV, decreased cell respiration and raised superoxide levels. Acute depletion of mitochondrial topoisomerase I triggered neither a nuclear mito-biogenic stress response nor compensatory topoisomerase IIβ upregulation, suggesting the concomitant increase in mitochondrial transcripts was due to release of a local inhibitory effect. Mitochondrial topoisomerase I was co-immunoprecipitated with mitochondrial RNA polymerase. It selectively accumulated and rapidly exchanged at a subset of nucleoids distinguished by the presence of newly synthesized RNA and/or mitochondrial RNA polymerase. The inactive Y559F-mutant behaved similarly without affecting mitochondrial transcripts. In conclusion, mitochondrial topoisomerase I dampens mitochondrial transcription and thereby alters respiratory capacity. The mechanism involves selective association of the active enzyme with transcriptionally active nucleoids and a direct interaction with mitochondrial RNA polymerase. The inhibitory role of topoisomerase I in mitochondrial transcription is strikingly different from the stimulatory role of topoisomerase I in nuclear transcription.

Highlights

Human mitochondrial DNA is a closed doublestranded DNA circle [1]
The increase was quantitatively similar for transcripts distributed across the entire length of heavy strand transcription (COX1, COX2, NADH dehydrogenase 2 (ND2), NADH dehydrogenase 5 (ND5)) or expressed under the control of the light strand promoter located in the displacement loop (D-loop) (ND6)
Levels of mitochondrial DNA (mtDNA) transcripts were normalized in TOP1MTÀ/À mouse embryonic fibroblasts (MEFs) on retroviral transduction of TOP1MT (Figure 1A) but not on transduction with virus particles containing the empty vector (Supplementary Figure S4), which confirms that the increases were caused by TOP1MT deficiency

Summary

Introduction

Strand separation during transcription and replication creates topological stress that interferes with mtDNA metabolism if not released by topoisomerases [2]. Two of the three topoisomerases so far found in mitochondria are produced from common genes encoding nuclear and mitochondrial enzyme variants. Mitochondrial topoisomerase IIIa (TOP3A) is produced by alternative translation initiation from a common transcript [3]. Mitochondrial topoisomerase IIb (TOP2B) is derived by limited proteolysis from the nuclear enzyme variety [4]. The exception is mitochondrial topoisomerase I (TOP1MT), which is encoded by a separate nuclear gene [5] conserved in vertebrates [6]. TOP1MTÀ/À mouse embryonic fibroblasts (MEFs) exhibit mitochondrial dysfunction and retrograde activation of a nuclear mito-biogenic stress response [7].

Methods

Results

Conclusion