Using sequencing to probe mtDNA transcription and replication

In a recent WCMR Science Seminar, we heard from Dr Katja Menger who’s research focus is investigating the role of new proteins involved in mitochondrial DNA replication and distribution. Here, Katja tells us more.

Dr Katja Menger

Human cells contain two forms of DNA. The vast majority of a cell’s DNA is located in the nucleus of the cell. However, a small portion of DNA is located inside mitochondria (mtDNA). Like nuclear DNA, the information in mtDNA needs to be accessed, copied into a carrier molecule called mRNA and subsequently translated into a protein sequence. These two processes are called transcription and translation respectively. mtDNA also needs to be copied correctly (a process called replication) to allow for an even distribution of mtDNA across the mitochondrial network and the cell.

We have previously identified that a protein named topoisomerase 3a (TOP3A) is involved in the separation of mtDNA molecules at the end of replication, and that mutations in this protein can lead to mitochondrial disease. TOP3A, as well as a second protein called mitochondrial topoisomerase 1 (TOP1MT), is responsible for removing knots and coils (topological constraints) within mtDNA molecules that can occur during transcription and replication. By using cells that don’t have either or both of the topoisomerases, we can ask questions about how these knots and coils influence mtDNA transcription and replication.

Katja presenting at our first WCMR Away Day on Rare Disease Day 2023.

Sequencing of DNA or RNA has previously been used primarily to identify changes in the genetic information stored in DNA, or to identify genes that are different in two different conditions, such as treated and untreated cells. In some of our recent work, we have looked at how we can use various sequencing techniques to identify mechanisms and issues during mtDNA transcription and replication.

Using a sequencing method called RNAseq, we found that the absence of TOP3A and TOP1MT had very little effect on the level of transcripts that are encoded in nuclear DNA. However, we could see a clear effect on the transcript levels of mtDNA in the case where no TOP3A was present. As all of the mtDNA molecule is transcribed into mRNA from only two locations on the mtDNA, the introduction of knots and coils during transcription should lead to increased interference in transcription with increasing distance from the start point of transcription. We found this to be the case in the TOP3A-depleted samples, suggesting that TOP3A plays a role in removing topological constraints during mtDNA transcription.

In addition, we looked at what happens during mtDNA replication in the absence of either TOP3A or TOP1MT, or both, and found that a large part of the mtDNA (the major arc) is overrepresented in the sequencing results in the absence of TOP3A or both topoisomerases. This points to a scenario where mtDNA replication has proceeded through the major arc and then stalled, suggesting that TOP3A is needed for replication to proceed further.

To conclude, we have shown that the two mitochondrial topoisomerases can remove the knots and coils introduced into mtDNA during replication and transcription. We can use a number of different sequencing techniques to identify how these topological constraints hinder either the replication or transcription machinery, and from the results infer the mechanisms by which both processes proceed in human cells.

To find out more about Katja’s research, click here.