Review ArticleThe Jekyll and Hyde character of RNase H1 and its multiple roles in mitochondrial DNA metabolism
Section snippets
Mitochondrial genome organization and expression
RNase H1 is located in the nucleus and the mitochondria, the two compartments of the cell that contain DNA, and here I focus exclusively on its activities in the mitochondria. Those readers familiar with the basics of mitochondrial DNA (mtDNA) metabolism can jump to the next section, but for those outside the field it is essential to have a map of mtDNA to help to navigate the remainder of the article. Mammalian mtDNA comprises 16.5 kilobases of duplex DNA typically arranged in circles, the two
RNase H1 is essential for mitochondrial DNA maintenance
In the first years of the millennium there were next to no knockout mice that lacked a gene essential for mtDNA maintenance, but the major mtDNA packaging protein TFAM had been ablated in 1998 and shown to result in mtDNA depletion and embryonic lethality around 10 days post-coitum (dpc) [7]. The reason the embryos survive for several days is because of the large maternal contribution of mtDNA from the oocyte [8]. Thus, the TFAM knockout mouse provided a critical clue when Bob Crouch´s group
RNase H1 is required for primer processing during mtDNA replication
The controversy concerning the mechanisms of mtDNA replication in mammals [10,11] has tended to obscure the areas where there is agreement. There is a consensus that the predominant mechanism of replication in mammalian mitochondria in most cells and tissues is strand-asynchronous; i.e. that there is a prolonged delay between the initiation of leading and lagging strand DNA synthesis. There is also agreement that for strand-asynchronous replication, DNA synthesis starts in the control region at
Pathological mutations in human RNase H1 cause mitochondrial disease
Given that the principal effect of RNase H1 ablation is the loss of mtDNA in the mouse [9], it came as no surprise to learn that mutations in the human gene can cause mtDNA defects and disease. The genetics linking mutations in RNASEH1 to individuals with classical features of mitochondrial disease and deletions of mtDNA were compelling [23], although the effects of the mutant RNase H1 variants on the protein and mtDNA were less clear. The first report suggested there was virtually no RNase H1
RNase H1 and the removal of aberrant R-loops from mitochondrial DNA
R-loops can form during transcription through the RNA hybridizing back to the template behind the advancing RNA polymerase (Fig. 4A), and such R-loops are universally recognized as a threat to genome integrity [24,25]. RNase H1 is an obvious candidate for degrading such R-loops; and when pathological mutations in RNase H1 (e.g. a isoleucine substitution, V142I) were found to slow mtDNA replication, it was proposed that this might be a consequence of the mutant enzyme failing to remove R-loops
The two faces of RNase H1 and the implications for the transcript-dependent mechanism of mtDNA replication
We have proposed that mature transcripts, or ‘bootlaces’, underpin the strand-asynchronous mechanism of mtDNA replication (e.g. [6,14,32]). At first sight, there could be no greater threat to a mechanism of replication reliant on RNA/DNA hybrids than RNase H1. Therefore, it was not immediately apparent why an absence of RNase H1 should cause a replication stalling phenotype ([18], Fig. 5A). One radical idea that is compatible with all the existing data is that RNase H1 works in tandem with, or
RNase H1 and the mitochondrial R-loop
The recently identified mitochondrial R-loop has a number of potential roles in mtDNA metabolism, and is a major potential target of RNase H1 [4,5]. Its RNA strand corresponds to the first transcript of the bootlace mechanism of replication, and the R-loop could contribute to mtDNA attachment to the inner mitochondrial membrane via the control region [34], which is expected to be critical for the organization and segregation of mtDNA. Cells with V142I RNase H1 display defects in all these
RNase H1 regulation of mitochondrial DNA replication
Oxidation has been reported to inactivate RNase H1 via the formation of a disulfide bridge [35], suggesting the enzyme can be redox regulated. The straightforward inference is that oxidizing conditions will inactivate RNase H1 and thus inhibit mtDNA replication. Modulating the redox environment in cells with wild-type RNase H1 and versions lacking one or both cysteines that form the disulfide bridge to inactivate the enzyme could therefore prove informative. Furthermore, the apparent
Concluding remarks: RNase H1 in mitochondria - the Goldilocks Conundrum
Too much or too little RNase H1 is catastrophic for mtDNA maintenance [18,36], and as set out in this article the enzyme has multiple established, as well as putative, diametrically opposed roles in mtDNA metabolism (Fig. 5, Fig. 6). Thus, the enzyme needs not only to be tightly regulated at the level of expression, but also strictly controlled from the time it enters the mitochondria to the moment it is degraded. Further study of the pathological variants of RNase H1 still has much to offer;
Conflict of interest statement
The author declares that he has no conflict of interest.
Funding
The author is supported by the Carlos III Program of Health (Pl17/00380) and the Department of Health, País Vasco (2018111043; 2018222031).
Acknowledgements
Bob Crouch and Brad Holmes for the collaboration on the function of RNase H1 in mitochondria. Antonella Spinazzola and two anonymous reviewers for critical comments on the manuscript, and Howy Jacobs for enlightening discussions in 2015, which gave rise to one of the key ideas in this review.
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