Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner.

There is plenty of literature suggesting the existence of mitochondrial RNA import in metazoa (Kim et al. 2017). Personally, I am not so convinced by this and it seems only some unicellular eukaryotes and plants have mitochondrial RNA import (Schneider 2011). There was recently an excellent review from Gammage et al. where they discussed the possibility of mitochondrial RNA import in metazoan (Gammage et al. 2018).

From the point of view of mitochondrial RNA import, one of the most hotly debated RNAs is 5S rRNA, a well-known structural component of the cytosolic ribosome. Only some single cell eukaryotes still have it in their mitochondrial DNA (mtDNA) (Janouškovec et al. 2017) but other have lost it. Recent structures of the mitochondrial ribosome showed that it doesn’t possess a 5S rRNA (Greber et al. 2015) but the 5S rRNA has actually been replaced by mtDNA encoded tRNA, the type of which depends on the species in question (Rorbach et al. 2016). So, from the point-of-view of the mitochondrial ribosome, there is no reason to import 5S rRNA.

I collected below (most) of the publications studying 5S rRNA import into mitochondria.

Publication	Method	Result
Yoshionari et al. 1994	Northern using mitoplasts	5S rRNA in mitoplasts is protected by RNAase
Magalhães et al. 1998	Northern and RT-PCR of mitochondrial RNA	5S rRNA in mitoplasts is protected by RNAase
Entelis et al. 2001	Northern of mitochondrial RNA	Different version of 5S rRNA imported with different efficiencies
Smirnov et al. 2008	Northern of mitochondrial RNA	Different version of 5S rRNA imported with different efficiencies
Smirnov et al. 2010	Northern of mitochondrial RNA	5S rRNA has mitochondrial import factors
Smirnov et al. 2011	Northern of mitochondrial RNA	More 5S rRNA import into mitochondria in the presence of pre-mRpL18
Mercer et al. 2011	RNAseq (mitochondria vs mitoplasts)	5S rRNA mildly enriched in mitoplasts vs mitochondria
Comte et al. 2013	Northern of mitochondrial RNA	5S rRNA in mitoplasts is protected by RNAase
Towheed et al. 2014	Northern of mitochondrial RNA	5S rRNA in mitoplasts is protected by RNAase and not imported upon membrane depolarization (FCCP)
Loutre et al. 2018	Northern of mitochondrial RNA	5S rRNA enriched in mitoplasts

I won’t to go to the specifics of each paper but when going through all of them I would have liked to see a few important controls:

• Verification of the mitoplast (mitochondria without the outer membrane) purity on western using antibodies against mitochondrial outer membrane, intermembrane space, inner membrane and matrix proteins.
• Show that the cytosolic ribosomes are not anymore attached to the mitochondrial outer membrane.
• Show that the enriched mitochondria are not contaminated by microsomes or endoplasmic reticulum.
• A RNA control which is not imported into mitochondria but would be digested under the experimental RNAse treatment conditions.
• How the ectopic expression of 5S rRNA or its derivatives affects cytosolic ribosome assembly/function and how much this contributing to mitochondrial changes.

The fact that almost all published mitochondrial 5S rRNA studies come from one lab using always the same approach make me a bit wary. The one published study using RNAseq suggested that 5S rRNA is enriched in mitochondria (Mercer et al. 2011) but some unpublished work from Vamsi Mootha’s lab using similar approach suggests 5S rRNA is just a persistent contaminant in mitochondrial preparations (Wolf 2014).

There was recently a publication suggesting that modified 5S rRNA can targeted into mitochondria and function as an anti-replicative molecule to stop the replication of mtDNA molecule carrying a deletions (Loutre et al. 2018). Figure 3 of this paper immediately raises some concerns as one of the negative control, the cytosolic 5.8S rRNA is also among the mitochondrial RNAs. The authors conclude this amount of contamination to be negligible. Next the authors established cell lines with stable expression of 5S rRNA and its variants. It seems weird to me that they never tested how this ectopic expression affects the main function of 5S rRNA, that is the cytosolic translation. I’m just worried that all the effects they detect are secondary to the changes in cytosolic translation.

These cell lines expressing 5S rRNA had also a heteroplasmic mtDNA deletion and the authors wanted to see whether 5S rRNA expression would affect the heteroplasmy levels of this deletion. Well, 5S rRNA and its derivatives had no effect on the heteroplasmy level of mtDNA deletion in these cell lines under normal glucose but showed some changes in glucose-free media. It should be pointed out though, that they carried out multiple comparisons using Student’s t-test so I wouldn’t trust the results.

Some cell clones with stronger ectopic expression of 5S RNA variants did show decrease in the mtDNA deletion levels. However, this could be also caused by the fact that cytosolic translation is affecting thereby affecting the whole cellular function. Again, they did multiple comparisons using Student’s t-test to analyze these results.

All in all, I find the evidence for mitochondrial 5S rRNA weak at best and people should start controlling better how ectopic 5S rRNA expression affects cytosolic translation. Otherwise everything they see might just be secondary to these cytosolic effects.

Reference:

Comte C, Tonin Y, Heckel-Mager AM, Boucheham A, Smirnov A, Auré K, Lombès A, Martin RP, Entelis N, Tarassov I. Mitochondrial targeting of recombinant RNAs modulates the level of a heteroplasmic mutation in human mitochondrial DNA associated with Kearns Sayre Syndrome. Nucleic Acids Res. 2013. PMID: 23087375

Entelis NS, Kolesnikova OA, Dogan S, Martin RP, Tarassov IA. 5 S rRNA and tRNA import into human mitochondria. Comparison of in vitro requirements. J Biol Chem. 2001. PMID: 11551911

Gammage PA, Moraes CT, Minczuk M. Mitochondrial Genome Engineering: The Revolution May Not Be CRISPR-Ized. Trends Genet. 2017. PMID: 29179920

Greber BJ, Bieri P, Leibundgut M, Leitner A2 Aebersold R, Boehringer D, Ban N. Ribosome. The complete structure of the 55S mammalian mitochondrial ribosome. Science. 2015. PMID: 25837512

Kim KM, Noh JH, Abdelmohsen K, Gorospe M. Mitochondrial noncoding RNA transport. BMB Rep. 2017. PMID: 28115039

Loutre R, Heckel AM, Jeandard D, Tarassov I, Entelis N. Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner. PLoS One. 2018. PMID: 29912984

Magalhães PJ, Andreu AL, Schon EA. Evidence for the presence of 5S rRNA in mammalian mitochondria. Mol Biol Cell. 1998. PMID: 9725900

Mercer TR, Neph S, Dinger ME, Crawford J, Smith MA, Shearwood AM, Haugen E, Bracken CP, Rackham O, Stamatoyannopoulos JA, Filipovska A, Mattick JS. The human mitochondrial transcriptome. Cell. 2011. PMID: 21854988

Rorbach J, Gao F, Powell CA, D'Souza A, Lightowlers RN, Minczuk M, Chrzanowska-Lightowlers ZM. Human mitochondrial ribosomes can switch their structural RNA composition. Proc Natl Acad Sci U S A. 2016. PMID: 27729525

Schneider A. Mitochondrial tRNA import and its consequences for mitochondrial translation. Annu Rev Biochem. 2011. PMID: 21417719

Smirnov A, Tarassov I, Mager-Heckel AM, Letzelter M, Martin RP, Krasheninnikov IA, Entelis N. Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria. RNA. 2008. PMID: 18314502

Smirnov A, Comte C, Mager-Heckel AM, Addis V, Krasheninnikov IA, Martin RP, Entelis N, Tarassov I. Mitochondrial enzyme rhodanese is essential for 5 S ribosomal RNA import into human mitochondria. J Biol Chem. 2010. PMID: 20663881

Smirnov A, Entelis N, Martin RP, Tarassov I. Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18. Genes Dev. 2011. PMID: 21685364

Towheed A, Markantone DM, Crain AT, Celotto AM, Palladino MJ. Small mitochondrial-targeted RNAs modulate endogenous mitochondrial protein expression in vivo. Neurobiol Dis. 2014. PMID: 24807207

Wolf AR. Leveraging genomic approaches to characterize mitochondrial RNA biology. Doctoral dissertation, Harvard University.

Yoshionari S, Koike T, Yokogawa T, Nishikawa K, Ueda T, Miura K, Watanabe K. Existence of nuclear-encoded 5S-rRNA in bovine mitochondria. FEBS Lett. 1994. PMID: 7508404

The mitochondrial DNA polymerase gamma degrades linear DNA fragments precluding the formation of deletions.

The mitochondrial DNA polymerase gamma degrades linear DNA fragments precluding the formation of deletions.

I posted recently how two labs have published data showing that the mitochondrial DNA  (mtDNA) polymerase gamma (POLGA) would degrade linear mtDNA fragments (Peeva et al. 2018) or mtDNA under starvation (Medeiros et al. 2018). Now a third group published similar results and they come from the lab of Carlos Moraes so you know are going to enjoy it (Nissanka et al. 2018).

In this study they used both mouse embryonic fibroblasts (MEFs) and actual mice. First, they expressed mitochondrially targeted restriction enzymes to produce linear mtDNA fragments and followed how quickly these are degraded in WT MEFs and mtDNA mutator MEFs. Clearly, MEFs expressing the exonuclease-deficient POLGA were not efficient in removing the linear mtDNA fragment which is a similar result to the Peeva et al. study.

Previously, Medeiros et al. and Peeva et al studied the degradation of mtDNA in yeast and cell culture, respectively, but it was unclear whether this would also take place in vivo. Moraes lab is rather experienced in introducing enzymes (restriction enzymes and mitoTALENs) into mitochondria in mice and as could be expected they introduced these mitochondrially targeted restriction enzymes into mice using adenovirus to study mtDNA degradation. As a result, it seems that POLGA is participating to the degradation of linear mtDNA fragments also in vivo.

Nissanka et al. also assessed whether the presence of linear mtDNA leads to mtDNA rearrangements such as circular mtDNA molecules with deletions. This seems to indeed be the case and it would be interesting to know whether these rearrangements would also take place in patients carrying pathogenic mutations in the replication machinery proteins. These results also suggest that mitochondrial zinc fingers (mtZFN) and mitoTALENs (Gammage et al. 2017) might have some unintended consequences. Both of these approaches are based on cutting the mtDNA molecules carrying a pathogenic mutation leading to the degradation of the molecule. In the ideal case, the loss of these pathogenic molecules would be replaced by the replication of the WT mtDNA molecules. Based on the results of Nissanka et al. in the non-ideal case the presence of these linear molecules could increase the amount of mtDNA rearrangements.

References:

Medeiros TC, Thomas RL, Ghillebert R, Graef M. Autophagy balances mtDNA synthesis and degradation by DNA polymerase POLG during starvation. J Cell Biol. 2018. PMID: 29519802

Nissanka N, Bacman SR, Plastini MJ, Moraes CT. The mitochondrial DNA polymerase gamma degrades linear DNA fragments precluding the formation of deletions. Nat Commun. 2018. PMID: 29950568

Gammage PA, Moraes CT, Minczuk M. Mitochondrial Genome Engineering: The Revolution May Not Be CRISPR-Ized. Trends Genet. 2017. PMID: 29179920

Peeva V, Blei D, Trombly G, Corsi S, Szukszto MJ, Rebelo-Guiomar P, Gammage PA, Kudin AP, Becker C, Altmüller J, Minczuk M, Zsurka G, Kunz WS. Linear mitochondrial DNA is rapidly degraded by components of the replication machinery. Nat Commun. 2018. PMID: 29712893

Impact of exercise on oocyte quality in the POLG mitochondrial DNA mutator mouse.

Mitochondrial DNA (mtDNA) mutator mouse carries a proofreading-deficient mitochondrial DNA polymerase leading to accumulation of mtDNA mutations. This mouse was originally engineered in two independent labs (Trifunovic et al. 2004, Kujoth et al. 2005) and has since been used to study various aspects of mitochondrial dysfunction.

Several studies have tried to find ways to improve the various phenotypes of the mtDNA mutator mouse, which include sarcopenia, hearing loss, osteoporosis, alopecia, weight loss, testicular atrophy, enlarged heart etc. For instance, our group showed recently that increasing mtDNA copy number of mtDNA mutator mice can partially rescue the testicular atrophy phenotype (Jiang et al. 2017)

It was published already seven years ago that exercise might improve the phenotype of the mitochondrial DNA (mtDNA) mutator mouse (Safdar et al. 2011). Like most papers using mtDNA mutator mouse as the model organism, Safdar et al. has the shortcoming that their WT control mice also inherited mtDNA mutations from a heterozygous mtDNA mutator mother, a.k.a. they made a “dirty” breeding (Kauppila et al. 2017). Therefore, this study had no “true” WT mouse model as a control. Also more recently, two papers from these authors have now Editorial Expression of Concerns (JBC study, PNAS study) so let’s see what happens with these findings.

Now a recent paper from a different group suggests that exercising mtDNA mutator mice might affect oocyte quality (Faraci et al. 2018). It was not reported how these mice have been bred, so one should be very cautious when interpreting the results. For instance, it would be important to know how many generations the heterozygous mtDNA mutator females have been bred and whether the WT control mice are a WT littermate from a heterozygote X heterozygote cross.

All in all, exercise has almost no effect on the oocytes of mtDNA mutator mice. For some of the differences seen it is also impossible to tell whether they are caused by changes in oocytes themselves of some systemic changes.

References:

Editorial Expression of Concern: Endurance exercise rescues progeroid aging and induces systemic mitochondrial rejuvenation in mtDNA mutator mice. Proc Natl Acad Sci U S A. 2018. PMID: 29891666

Expression of concern for: Exercise increases mitochondrial PGC-1 α content and promotes nuclear-mitochondrial cross-talk to coordinate mitochondrial biogenesis. J Biol Chem. 2018. PMID: 29602880

Faraci C, Annis S, Jin J, Li H, Khrapko K, Woods D. Impact of exercise on oocyte quality in the POLG mitochondrial DNA mutator mouse. Reproduction. 2018. PMID: 29875308

Jiang M, Kauppila TES, Motori E, Li X, Atanassov I, Folz-Donahue K, Bonekamp NA, Albarran-Gutierrez S, Stewart JB, Larsson NG. Increased Total mtDNA Copy Number Cures Male Infertility Despite Unaltered mtDNA Mutation Load. Cell Metab. 2017. PMID: 28768180

Kauppila TES, Kauppila JHK1, Larsson NG. Mammalian Mitochondria and Aging: An Update. Cell Metab. 2017. PMID: 28094012

Kujoth GC, Hiona A, Pugh TD, Someya S, Panzer K, Wohlgemuth SE, Hofer T, Seo AY, Sullivan R, Jobling WA, Morrow JD, Van Remmen H, Sedivy JM, Yamasoba T, Tanokura M, Weindruch R, Leeuwenburgh C, Prolla TA. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science. 2005. PMID: 16020738

Safdar A, Bourgeois JM, Ogborn DI, Little JP, Hettinga BP, Akhtar M, Thompson JE, Melov S, Mocellin NJ, Kujoth GC, Prolla TA, Tarnopolsky MA. Endurance exercise rescues progeroid aging and induces systemic mitochondrial rejuvenation in mtDNA mutator mice. Proc Natl Acad Sci U S A. 2011. PMID: 21368114

Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, Bruder CE, Bohlooly-Y M, Gidlöf S, Oldfors A, Wibom R, Törnell J, Jacobs HT, Larsson NG. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature. 2004. PMID: 15164064