Fixing mitochindrial mutations with targeted mitochondrial RNA import

If you've been reading the blog for a while now, you've heard this many times: not all mutations are deleterious, but the ones that are can increase the risk of certain cancers and diseases. Numerous genetic defects have been attributed to mutations, and mutation in the mtDNA, the mitochondrial DNA, are no exceptions:

"Specific mutations in mtDNA have been implicated in muscular and neuronal disease and in the decline of organ function with aging. Despite a significant need, there are currently no effective treatments for mtDNA alterations [1]".

I've often talked about gene therapy as a way to try and fix defects caused by genetic mutations. The idea behind gene therapy is to introduce the non-mutated gene into the cells so that the production of the healthy protein can be restored. So, a natural question to ask would be: can we use gene therapy to fix mtDNA mutations as well?

Unfortunately the answer is no: if a nuclear encoded protein is mutated, it may fail to assemble correctly in the cytosol and thus it may be prevented from entering the mitochondria correctly.  Fortunately, though, Wang et al. [1] had a different idea.

Back in February I discussed the migration of genes from the organelles to the nucleus and, in some cases, back to the nucleus, a phenomenon that took place throughout evolution. One of the consequences is that in the human genome the majority of mitochondrial proteins are encoded in nuclear genes. I recently discovered that this import/export activity is also true for RNA, tRNAs in particular.

Transfer RNA, or tRNA, is the type of RNA that pairs a complementary triplet (anti-codon) with each of the mRNA coding triplets (codon) as specified by the set of rules of the genetic code. This helps to place the corresponding amino acid into the right position of the protein sequence, as illustrated in the following figure from Wikipedia.

In their March PNAS paper, Wang et al. note:

"A subset of nucleus-encoded tRNAs is imported into the mitochondria of almost every organism. The number of imported tRNAs ranges from one in yeast to all in trypanosomes [protozoa] [. . .] The nucleus-encoded RNAs in the mitochondrion have potentially diverse import pathways, but the details of these pathways and import mechanisms are still being revealed."

Wang and colleagues asked the following question: could we use this naturally happening import activity to import healthy RNA into the mitochondria and "fix" mitochondrial mutations?

First, they made sure that the tRNAs they were going to use to "fix" the mutations were correctly imported into the mitochondria. In order to do so, the researchers appended a sequence, called RP sequence, to the 5' end of the RNAs. They observed, both in vitro and in vivo in mouse models, that

"Only mt-RNA precursors with the appended RP sequence were efficiently imported into isolated mitochondria."

In vivo, they generated expressions of the hCOX2 gene, with and without the RP import sequence, and introduced them in mouse cells.

"Cells expressing RP-hCOX2, but not hCOX2, nucleus-encoded mtRNA showed mitochondrial transcript import and hCOX2 protein translation within mitochondria, indicating that the RP import sequence also is required and functions with coding mtRNA in vivo."

Summarizing, the RP sequence functions as a "driver" that directs the RNA into the mitochondria and, once there, the RNA is indeed translated and the corresponding protein expressed.

Wang et al. applied all of the above to a cell line that harbors two mutations. Both mutations cause an inefficiency in tRNA translation, which results in defective cell respiration. Remember, tRNA is what defines the protein primary structure out of the "instructions" handed over by the mRNA. So, if there's not enough tRNA, not enough proteins are made, and that usually isn't a good thing. Though a complete recovery was not expected, as the mutant mt-tRNAs are still present, not substituted, the imported wild-type mt-tRNAs with three elements expressed (the extended stem, the RP sequence, and MRPS12 3' UTR -- see paper for details) were able to partly restore the respiratory defect caused by the mutations.

Compared to previous methods, this approach is innovative because it doesn't require the introduction of non-native TRNAs with foreign protein factors.

"Rationally engineered human mitochondrial tRNAs and mRNAs can both be efficiently targeted and functional. The fusion RNA presequences are encoded in the nuclear genome and can be imported into mitochondria where they are processed, restore translation, and are degraded via normal pathways in the mitochondrion."

[1] Wang, G., Shimada, E., Zhang, J., Hong, J., Smith, G., Teitell, M., & Koehler, C. (2012). Correcting human mitochondrial mutations with targeted RNA import Proceedings of the National Academy of Sciences, 109 (13), 4840-4845 DOI: 10.1073/pnas.1116792109

Is cancer contagious? Sometimes. But it may not be a bad thing.

About 15% of all cancers worldwide are caused by infectious pathogens such as viruses, bacteria, or parasites [1]. Viruses that are capable of inducing cancer are called oncoviruses -- HPV is an example. The pathogen is transmitted from a donor to a recipient, starts the infection, and the infection eventually causes the cancer. But did you know there existed such a thing as a transmissible cancer? In this case, it's not the pathogen, but the cancer cell line itself that gets transmitted from one individual to another.

Yes, it's scary, but there are some good news.

For one thing, "relatively common" cases have been observed in animals only. Canine transmissible venereal tumor (CTVT) is quite common in dogs. It's transmitted during mating and eventually rejected by the host dog who then acquires lifelong immunity.

"In man, only scattered case reports exist about such communicable cancers, most often in the setting of organ or hematopoietic stem cell transplants and cancers arising during pregnancy that are transmitted to the fetus. In about one third of cases, transplant recipients develop cancers from donor organs from individuals who were found to harbor malignancies after the transplantation. The fact that two thirds of the time cancer does not develop, along with the fact that cancer very rarely is transmitted from person to person, supports the notion that natural immunity prevents such cancers from taking hold in man. These observations might hold invaluable clues to the immunobiology and possible immunotherapy of cancer [1]."

CTVT is particularly interesting to study because it has evolved some ingenious mechanisms to escape the immune system. Every nucleated cell has a class of molecules, called MHC, which have the function to display fragments of proteins that are "flags" as to whether the cell is healthy or harbors some pathogen. Once in the host, CTVT

"downregulates its MHC I expression, thereby reducing its initial visibility to the host's immune system. This allows it to not only to escape T-cell mediated immunity (which would occur if MHC I were fully expressed) but also natural killer cells (which would eradicate the cells were they completely devoid of MHC I)."

Despite this type of "defense", eventually the dog's immune system recognizes the pathogen and clears it, and understanding this mechanism is of interest for a possible cancer vaccine (I talked about cancer vaccines here).

A recent study published in Science [2] looked at two regions in the mitochondrial genome (mtDNA) from 37 CTVT samples, and compared them with sequences from the mtDNA of 15 hosts. Through phylogenetic analysis Rebbeck et al. showed a high variability in the sequenced regions, suggesting that CTVT periodically acquires mtDNA from infected hosts. The reason for this, the researchers hypothesize, is that CTVT mitochondria, due to a high metabolic rate, tend to accumulate deleterious mutations and therefore, transfers of mtDNA from the host may have the benefit of restoring CTVT mitochondrial function.

[1] Welsh, J. (2011). Contagious Cancer The Oncologist, 16 (1), 1-4 DOI: 10.1634/theoncologist.2010-0301

[2] Rebbeck, C., Leroi, A., & Burt, A. (2011). Mitochondrial Capture by a Transmissible Cancer Science, 331 (6015), 303-303 DOI: 10.1126/science.1197696

The "not-so-universal" genetic code, its origin and its evolution

From [1]:

"Until relatively recently, the [genetic] code was thought to be invariable, frozen, in all organisms, because of the way in which any change would produce widespread alteration in the amino acid sequences of proteins. The universality of the genetic code was first challenged in 1979, when mammalian mitochondria were found to use a code that deviated somewhat from the universal."

A brief refresher: proteins are chains of amino acids. They are made from messenger RNA by assigning each triplet of RNA nucleotides (a codon) to one amino acid. For example, in the sequence AUGCCCAAGCUG each triplet codes an amino acid: AUG becomes M, CCC becomes P, AAG becomes K, and CUG becomes L. All together: AUG|CCC|AAG|CUG -> MPKL.

So, what does "universal" mean in the above quote? It means that the above sequence gets translated into the same amino acids in every organism, from bacteria to humans. Is this true? Not always.

Take a stop codon, for example. A stop codon is a triplet of RNA nucleotides that end the translation. Think of it as a flag that says, "The protein code ends here." If the genetic code were a universal one, a stop codon would always be a stop codon, in all organisms. The first exception to this was discovered in 1985, when the stop codon UGA was found to be actually coding an amino acid in the bacteria Mycoplasma capricolum. More exceptions to the "universal" conception (other triplets that coded different amino acids instead of always the same one) were later found in other organisms and in mitochondrial DNA as well. A more realistic theory is that, being DNA dynamical, when codons "disappear" the old codons can undergo reassignments and take on a new meaning.

The "universal" view has prevailed for many years on the basis that present time proteins are so evolved that changes would most likely be lethal. The first deviations from universality were found in the late 'seventies in mitochondrial DNA. It was argued that mtDNA is considerably smaller than nuclear DNA and hence it had a better tolerance to changes.

In [1], Ohama et al. list various code changes reported in the nuclear DNA in the past three decades, and then discuss the origin of the genetic code:

"The theories to explain the early evolution of the genetic code are numerous, all of which include speculations that the coding system arose with one or a limited number of amino acids, and that others were added until a total of 20 was reached. Most of these theories are aesthetically pleasing but cannot be verified."

They assume that the most ancient genetic code had to have a minimum number of codons made of all 20 amino acids and a minimum number of corresponding tRNAs -- transfer RNA molecules that act as mediators between the mRNA and the amino acids. This first genetic code had to have very little tolerance for change. However, with the time, the development of synonymous codons (different triplets code the same amino acid), allowed for flexibility and therefore resulted in an advantageous addition.

Finally, they conclude:

"It should be stressed however that there are no organisms which use the genetic code system for more than, or less than, 20 amino acids. What were frozen are 20 amino acids (magic 20!) and not the genetic code that assigns them. Thus the genetic code is still in the state of evolution."

I'm including below a second reference [2] that goes a bit more in depth on how these codon reassignments happen, for those of you who might be interested. In this case, the authors looked at the evolution of the genetic code in yeast.

[1] Ohama T, Inagaki Y, Bessho Y, & Osawa S (2008). Evolving genetic code. Proceedings of the Japan Academy. Series B, Physical and biological sciences, 84 (2), 58-74 PMID: 18941287

[2] Miranda, I., Silva, R., & Santos, M. (2006). Evolution of the genetic code in yeasts Yeast, 23 (3), 203-213 DOI: 10.1002/yea.1350

Migrating genes

I've been talking quite a lot about mitochondria lately. The fact that these organelles contain their own DNA (called mtDNA) and were the result of a horizontal gene transfer during evolution is simply fascinating. And I know many of you agree, as proved by the wonderful questions my last post on mitochondria sparked (thank you Hollis and Marleen!)

Plastids, plant organelles that are responsible for photosynthesis, also have a circular, double-stranded DNA molecule (called ptDNA). Like mitochondria, plastids originated through endosymbiosis and, in most plants, are inherited from one parent only. Now, here's another fascinating fact:

"During the early phase of organelle evolution, organelle-to-nucleus DNA transfer resulted in a massive relocation of functional genes to the nucleus: in yeast, as many as 75% of all nuclear genes could derive from protomitochondria, whereas ~4500 genes in the nucleus of Arabidopsis are of plastid descent. Cases of present-day organelle-to-nucleus DNA transfer, revealed by the presence of NUMTs and NUPTs [the fraction of nuclear DNA that derives from mitochondria and plastids respectively], are known in most species studied so far. [...] Mitochondrial chromosomes contain segments homologous to chloroplast sequences, as well as sequences of nuclear origin, providing indirect evidence for plastid-to-mitochondrion and nucleus-to-mitochondrion transfer of DNA [1]."

Throughout evolution transfers of genes between plastids and mitochondria have been documented, although in present organisms these transfers gave rise to non-functional sequences. In plants, the transfer of genes from organelles to nucleus seems to be still active, as documented by the RPS10 gene, which is present in the mitochondria of some angiosperms, and in the nucleus of other plants [Henze and Martin, 2001]. In fact, orgenelle-to-DNA gene transfers have been studied extensively in plants: their cells have both plastids and mitochondria and, as a consequence, they are in general more informative than animal eukaryotes.

In [1], Leister revises studies that show that mtDNA in Homo sapiens integrates continuously into the nuclear genome, as both de novo and pre-existing nuclear insertions of mtDNA have been documented. Recent acquisition of nuclear mtDNA have been documented by comparison with chimpanzee genomes.

So, how do these transfers happen? Though it was originally thought that genes would migrate as RNA transcripts, new studies have shown that it's the DNA itself that "escapes" the organelle:

"Escape of organelle DNA and its uptake into the nucleus has been experimentally demonstrated in yeast and tobacco."

Once these bits of DNA arrive to the nucleus, however, they are subject to a much lower mutation rate than they were in their original location. What this means is that mutations appear more rarely in the nucleus than they do in the organelles. As a consequence, they become "conserved," undergo very little changes, and, at all effects, become "molecular fossils," allowing researchers to retrace phylogenies between species.

"Moreover, nuclear organelle DNA insertion polymorphisms, as a subclass of insertion-deletion polymorphisms, are valuable markers for population and evolutionary studies."

Since the process of migration from organelle to nucleus is a constant one, studies have been directed at measuring the rate of continued colonization of organelle DNA into the germline. The rate in humans has been found to be of the order of 10e-05, and even though these insertions in the past had been thought to be essentially harmless, recent studies have confirmed associations with certain types of hereditary diseases. As I was discussing last week, more studies are in the way to investigate possible associations between nuclear mitochondrial polymorphisms and certain types of cancers.

[1] Leister, D. (2005). Origin, evolution and genetic effects of nuclear insertions of organelle DNA Trends in Genetics, 21 (12), 655-663 DOI: 10.1016/j.tig.2005.09.004

The wondrous mitochondrion and its proteome

The sequencing of human mitochondrial DNA, a circular DNA molecule contained in mitochondria, was completed in 1981, and, since then, roughly 150 mutations have been found that are associated with maternally inherited diseases (if you don't remember why mtDNA is inherited from the mother and not the father, check out this earlier post of mine). Despite this, the majority of human mitochondrial syndromes are actually caused by defects in the nuclear genome. This is sort of obvious if you think about it, given that human mitochondrial proteome consists of an estimated 1,100-1,400 distinct proteins, of which 13 are encoded by the mitochondrial DNA. The majority of proteins targeted at the mitochondria are actually encoded by nuclear genes. In fact

"The 13 proteins encoded by mammalian mtDNA are all components of the respiratory chain, which generates the majority of cellular ATP via oxidative phosphorylation (OXPHOS). However, the remaining respiratory chain subunits are encoded by nuclear genes, as are all proteins required for the transcription, translation, modification, and assembly of the 13 mtDNA proteins. All the components of numerous other mitochondrial pathways are also nuclear en- coded, including the tricarboxylic acid (TCA) cycle, protein import, fatty acid and amino acid oxidation, apoptosis, and biosynthesis of ketone bodies, pyrimidines, heme, and urea. Furthermore, during the decades following the sequencing of the mtDNA, it became clear that maternally inherited mitochondrial disorders represent only 20% of all inherited human mitochondrial disorders [1]."

Mitochondria are amazing organelles. Roughly half of mitochondrial proteins are ubiquitous and found across all organs, while the rest are tissue specific, meaning that their function and structure varies across cell lines. For example, when comparing mitochondria across different tissues, researchers found about a 75% overlap. In addition to this cell-type specificity, some of the mitochondrial proteins are expressed at very low levels or only during certain specific developmental stages, making the characterization of the mitochondrial proteome a challenging task. Today, a little over 1,000 of all mitochondrial proteins have been identified, mainly through large-scale proteomics, microscopy, and computation.

The first half of Calvo and Mootha's review [1] is an detailed report on the progress made so far in extensively classifying the mitochondrial proteome. They then proceed to discuss how the inventory of mitochondrial proteins has lead to the better understanding of mitochondrial disorders as well as the discovery of new disease genes.

"Traditionally, mitochondrial disease has referred primarily to disorders of oxidative ATP production, as discussed above. However the breadth of the mitochondrial proteome now implicates a large number of additional phenotypes, such as soft tissue tumors (paragangliomas) and diabetes mellitus. The discovery of new disease genes will further expand the clinical phenotypes associated with mitochondrial defects."

[1] Calvo, S., & Mootha, V. (2010). The Mitochondrial Proteome and Human Disease Annual Review of Genomics and Human Genetics, 11 (1), 25-44 DOI: 10.1146/annurev-genom-082509-141720

Another genetic puzzle: why is mitochondrial DNA only inherited from the mother's side?

Remember when I told you that bacteria have circular DNA? Well, we have it too, only not in the nucleus where the rest of our DNA sits. It's a rather interesting story, one that biologist Lynn Margulis proved in 1967 [1]: our cells contain organelles called mitochondria, which originally were separate organisms (prokaryotes), and at some point entered a symbiotic relationship with eukaryotic cells through endosymbiosis. As a result, they contain their own, circular DNA called mitochondrial DNA or, in short, mtDNA.

Circularity is not the only fascinating thing about mtDNA. It contains 37 genes, and, because it's not found in the nucleus, non-nucleated cells like precortical cells (found in hair shafts) can't be used for DNA analysis, but can indeed be used to extract mtDNA. However, whereas nuclear DNA is unique to each individual, mtDNA is not. That's because it's inherited exclusively through the maternal lineage. As you know, paternal and maternal chromosomes undergo recombination and then fuse together to make the unique DNA of a new individual. However, mtDNA does not undergo recombination and the only variation happening is due to random mutations when the cell splits. These are quite rare and in fact, it's not unusual to share identical mtDNA with our siblings, and/or to inherit it unchanged from our mothers.

Maternal mtDNA inheritance occurs in most eukaryotic species, indicating that, from an evolutionary point of view, it's an old and conserved mechanism. One might argue that paternal gametes (sperm) are much smaller than maternal gametes (eggs) and therefore contribute a limited amount of mitochondria, which then get lost. In fact, the general belief was that, at least in some species, paternal mitochondria was excluded due to the fact that only the head of the spermatozoon enters the oocyte's cytoplasm. Well, that doesn't quite explain the whole story, and the mechanism that allows the clearance of paternal mitochondria during early embryonegesis was not understood until recently.

Two studies published in the November 25 issue of Science [2, 3] used a Caenorhabditis elegans model to show that the degradation of paternal mitochondria is achieved through involvement of autophagosomes, double membrane vesicles that recruit the organelles, engulf them, and then destroy them. Rawi et al. also proved that autophagy is triggered in the mouse too, within minutes after fertilization, whereas, in the absence of autophagosomes (which they induced artificially in some animals), the paternal mitochondria persist in the embryos.

This is a great step forward, but many questions remain unanswered, as Levine and Elazar note in the accompanying perspective:

"The findings of Sato and Sato and Al Rawi et al. help to explain how paternal mitochondria and mtDNA are destroyed, but why they are destroyed remains a mystery. Is heteroplasmy, the occurrence of more than one mtDNA genotype, dangerous for the developing embryo? Or is the degradation of paternal mitochondria merely a primitive defense in which the fertilized oocyte views the paternal mitochondria as a potentially dangerous intruder that must be destroyed?"

[1] Sagan, L. (Margulis, L.) (1967). On the origin of mitosing cells Journal of Theoretical Biology, 14 (3) DOI: 10.1016/0022-5193(67)90079-3

[2] Sato, M., & Sato, K. (2011). Degradation of Paternal Mitochondria by Fertilization-Triggered Autophagy in C. elegans Embryos Science, 334 (6059), 1141-1144 DOI: 10.1126/science.1210333

[3] Al Rawi, S., Louvet-Vallee, S., Djeddi, A., Sachse, M., Culetto, E., Hajjar, C., Boyd, L., Legouis, R., & Galy, V. (2011). Postfertilization Autophagy of Sperm Organelles Prevents Paternal Mitochondrial DNA Transmission Science, 334 (6059), 1144-1147 DOI: 10.1126/science.1211878