Numt

NUMT, pronounced 'new might,' is an acronym for 'nuclear mitochondrial DNA' segment coined by evolutionary geneticist, Jose V. Lopez, which describes a transposition of any type of cytoplasmic mitochondrial DNA into the nuclear genome of eukaryotic organisms. NUMT, pronounced 'new might,' is an acronym for 'nuclear mitochondrial DNA' segment coined by evolutionary geneticist, Jose V. Lopez, which describes a transposition of any type of cytoplasmic mitochondrial DNA into the nuclear genome of eukaryotic organisms. More and more NUMT sequences, with different size and length, in the diverse number of Eukaryotes, have been detected as more whole genome sequencing of different organisms accumulate. In fact, NUMTs have often been unintentionally discovered by researchers who were looking for mtDNA. NUMTs have been reported in all studied eukaryotes, and nearly all mitochondrial genome regions can be integrated into the nuclear genome. However, NUMTs differ in number and size across different species. Such differences may be accounted for by interspecific variation in such factors as germline stability and mitochondria number.After the release of the mtDNA to the cytoplasm, due to the mitochondrial alteration and morphological changes, mtDNA is transferred into the nucleus by one of the various predicted methods and are eventually inserted by double-stranded break repair processes into the nuclear DNA (nDNA). Not only has any correlation been found between the fraction of noncoding DNA and NUMT abundance in the genome but NUMTs are also proven to have non-random distribution and a higher likelihood of being inserted in the certain location of genome compare to others. Depending on the location of the insertion, NUMTs might perturb the function of the genes. In addition, De novo integration of NUMT pseudogenes into the nuclear genome has an adverse effect in some cases, promoting various disorders and aging. The first application of the NUMT term in the domestic cat (Felis catus) example was striking, since mitochondrial gene number and content were amplified 38-76X in the cat nuclear genome, besides being transposed from the cytoplasm. The presence of NUMT fragments in the genome is not problematic in all species; for instance, it is shown that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. Although, the extended translocation of mtDNA fragments and their co-amplification with free mitochondrial DNA has been problematic in the diagnosis of mitochondrial disorders, in the study of population genetics, and phylogenetic analyses, scientists have used NUMTs as the genetic markers to figure out the relative rate of nuclear and mitochondrial mutation and recreating the evolutionary tree. Mitochondria, as a major energy factory of the cell, was previously a free-living prokaryote that invades the eukaryotic cells by the endosymbiosis theory, which gained acceptance around the 1970s. Under this theory, symbiotic organelles gradually transferred their genes to the eukaryotic genome, implying that mtDNA was gradually integrated into the nuclear genome. Despite the metabolic alterations and functional adaptations in the host eukaryotes, circular mitochondrial DNA is contained within the organelles. Containing 37 genes, mitochondrial DNA has an essential role in the production of necessary compounds, such as required enzymes for the proper function of mitochondria. Specifically, it has been suggested that certain genes (such as the genes for cytochrome oxidase subunits I and II) within the organelle are necessary to regulate redox balance throughout membrane-associated electron transport chains. These parts of the mitochondrial genome have been reported to be the most frequently employed. Mitochondria is not the only location within which the cell mtDNA, mitochondrial DNA, can be found; sometimes transfer of mitochondrial DNA from organelles to the nucleus can occur; the evidence of such translocation has been seen through the comparison of mitochondrial DNA sequence with the genome sequence of the counterparts. The integration and recombination of cytoplasmic mtDNA into the nuclear DNA is called Nuclear Mitochondrial DNA, which is abbreviated as NUMT.The possible presence of organelle DNA inside the nuclear genome was suggested after finding of homologous structure to the mitochondrial DNA, which was shortly after the discovery of the presence of an independent DNA within the organelles in 1967. This topic stayed untouched until the 1980s. Initial evidence that DNA could move among cell compartments came when fragments of chloroplast DNA were found in the maize mitochondrial genome with the help of cross-hybridization, between chloroplast and mitochondrial DNA, and physical mapping of homologous regions. After this initial observation, Ellis coined the name 'promiscuous DNA' in order to signify the transfer of DNA intracellularly from one organelle to the other and is the presence of organelle DNA in multiple cellular compartments. This is not only an important discovery on its own, but is also highly informative and helpful for understanding the evolutionary process and the time period different occurrence might take place. The searching for mtDNA in nuclear DNA continued until 1994 when the recent remarkable transposition of 7.9 kb of a typically 17.0-kb mitochondrial genome to a specific nuclear chromosomal position in the domestic cat was reported. This is the time that NUMT was coined to designate the large stretches of mitochondrial DNA in the genome.Up to now, the whole genomes of many eukaryotes, both vertebrate, and invertebrate, have been sequenced and NUMT was observed in the nuclear genome of various organisms, including yeast, Podospora, sea urchin, locust, honey bee, Tribolium, rat, maize, rice, and primates. In Plasmodium, Anopheles gambiae, and Aedes aegypti mosquitoes NUMT can barely be detected. In contrast, the conserved fragments of NUMT have now few were identified in genome data for Ciona intestinalis, Neurospora crassa, Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, and Rattus norvegicus. Antunes and Ramos were found the presence of NUMT in the fish genome for the first time in 2005 using of BLASTN, MAFFT, very vigorous genome mappings, and phylogenic analysis. Across the animal kingdom, Apis mellifera, from phylum Arthropoda, and Hydra magnipapillata, from phylum Cnidaria, are respectively the first and second animals with the highest ratio of NUMTs to the total size of the nuclear genome while Monodelphis Domestica, or Gray short-tailed opossum, is the record holder for NUMT frequency among vertebrates. Similar to animals, NUMTs are abundant in the plants and the longest NUMT fragment known so far, a 620-kb partially duplicated insertion of the 367-kb mtDNA of Arabidopsis thaliana, was reported. NUMT insertion into the nuclear genome and its persistence in the nuclear genome initiated by physical delivery of mitochondrial DNA to the nucleus. This step follows by the mtDNA integration into the genome through a non-homologous end joining mechanism during double-strand break (DSB) repair process as envisioned by studying baker's yeast, Saccharomyces Cerevisiae; and terminates by intragenomic dynamics of amplification, mutation, or deletion, which also known as post-insertion modifications. The mechanism of mtDNA transfer into nucleus has not yet fully understood. Transfer of the released mtDNA into the nucleus: The first step in the transferring process is the release of mtDNA into the cytoplasm. Thorsness and Fox demonstrated the rate of relocation of mtDNA from mitochondria into the nucleus using ura3- yeast strain with an engineered URA3 plasmid, required gene for uracil biosynthesis, in the mitochondria. During the propagation of such yeast strains carrying a nuclear ura3 mutation, plasmid DNA that escapes from the mitochondrion to the nucleus, complements the uracil biosynthetic defect, restoring growth in the absence of uracil, and easily scored phenotype. The rate of DNA transfer from the mitochondria to the nucleus was estimated as 2 x 10-5 per cell per generation while the opposite, in the case of cox2 mutant, the rate of the transfer of plasmid from the nucleus to the mitochondria is apparently at least 100,000 times less. Many factors control the rate of mtDNA escapes from mitochondria to the nucleus. The higher rate of mutation in mtDNA in comparison with nDNA in the cells of many organisms is an important factor promoting the transfer of mitochondrial genes into the nuclear genome. One of the intergenic factors results in the higher destruction rate of mitochondrial macromolecules, including mtDNA, is the presence of high level of reactive oxygen species (ROS), generated in mitochondria as the by-products in ATP synthesis mechanism. Some other factors influencing the escape of mtDNA from mitochondria include the action of mutagenic agents and other forms of cellular stress that can damage mitochondria or their membranes, which proves that is possible to assume that exogenous damaging agents (ionizing radiation and chemical genotoxic agents) increase the rate of mtDNA escape into the cytoplasm. Thorsness and Fox continued their research to find the endogenous factors affecting mtDNA escape into the nucleus. They isolated and studied 21 nuclear mutants with different combinations of mutations in at least 12 nuclear loci called the yme (yeast mitochondrial escape) mutations, in different environmental conditions since some of these mutations cause temperature sensitivity. They found out these mutations which perturb mitochondrial functions, due to the alteration of gene products, affect mitochondrial integrity and led to mtDNA escape to the cytoplasm. Additionally, defects in the proteins change the rate of mtDNA transfer into the nucleus. For instance, in the case of yme1 mutant, abnormal mitochondria are targeted for degradation by the vacuole, with the help of pep4 , a major proteinase, and degradation increases mtDNA escape to the nucleus through the process of mitophagy. In addition, Thorsness and Campbell found that by disruption of pep4, the frequency of mtDNA escape in yme1 strains decreases. Similarly, the disruption of PRC1, which encodes carboxypeptidase Y, lowers the rate of mtDNA escape in yme1 yeast. Evidence shows that mitophagy is one of the possible ways for mtDNA transfer into the nucleus and determined to be the most supported pathway up to now. Some other possible pathways are shown in figure 1. The first pathway, as it was explained, is a yme1mutant that results in inactivation of YMe1p protein, a mitochondrial-localized ATP-dependent metalloproteinase, leading to high escape rate of mtDNA to the nucleus. Mitochondria of yme1 strain are taken up for degradation by the vacuole more frequently than the wild-type strain. Moreover, cytological investigations have suggested several other possible pathways in the diverse number of species, including a lysis of the mitochondrial compartment, direct physical connection and membrane fusion between mitochondria and nucleus, and encapsulation of mitochondrial compartments inside the nucleus, as shown in figure 1. Pre-insertion preparation: After reaching the nucleus, mtDNA has to enter the nuclear genome. The rate of mtDNA incorporation into the nuclear genome can be expected to depend on the DSB number in nDNA, the activity of DSB repair systems, and the rate of mtDNA escape from organelles. MtDNA insertion comprises three main processes, shown in figure 2; first, the mtDNA has to have the proper form and sequence; in other words, the mtDNA has to be edited which gives a rise to the new edited site in the polynucleotide structure. Mitochondrial DNA is not universal and, in animals similar to plants, mitochondrial editing shows very erratic patterns of taxon-specific occurrence. As shown in figure 2, there are three possible ways that mtDNA can become prepared to be inserted into the nuclear DNA. The process mainly depends on the time mtDNA transfers into the nucleus. As shown in figure 2b, direct integration of unedited mtDNA fragments into the nuclear genomes is the most plausible and the evidence both found in plants, Arabidopsis genome, and animals with the help of different methods, including BLAST-based analysis. In this case, mtDNA is transferred into the nucleus whereby editing and introns arise in the mitochondrion later. If a gene, for instance, was transferred to the nucleus in one lineage before mitochondrial editing evolved, but remained in the organelle in other lineages where editing arose, the nuclear copy would appear more similar to an edited transcript than to the remaining mitochondrial copies at the edited sites. Another represented and less supported model, figure 2a, is the cDNA-mediated model, which intron-contained mtDNA enters the nucleus and by reverse transcription of spliced and edited mitochondrial transcript, it becomes integrated into the nDNA. The third proposed mechanism is the direct transfer and integration of intronless mtDNA into the nucleus, figure 2c, whereby editing and introns in the mitochondrion come and go during evolution. In this case, the introduction and removal of the intron, as well as, reverse transcription occur within mitochondria and the final product, the edited intronless mtDNA, will integrate into nDNA after being transferred into the nucleus. Insertion into the nuclear genome:After the preparatory step is over, mtDNA is ready to be inserted into the nuclear genome. Based on NUMT integration site and the analyzed obtained results from baker's yeast experiment, Blanchard and Schmidt hypothesized that mtDNA are inserted into the double-stranded break (DSB) via non-homologous end joining machinery. The hypothesis is found to be widely accepted. Later analyses were consistent with the involvement of NHEJ in NUMT integration in humans. These processes occur in both somatic and germline cells. In animals and humans, however, the capability of DSB repair in germline cells depends on the oogenetic and spermatogenetic stage, nonetheless, due the low repair activity, mature sperms are incapable of DSB repair. Additionally, DSB can also be repaired by homologous recombination (HR), which is more accurate and introduces fewer errors in the process of repair, while, has not yet seen in the process of mtDNA insertion;. Apart from canonical NHEJ, DSBs are repaired via a mechanism that involves sequences containing a few homologous nucleotides at the ends of a DSB to be ligated. This mechanism is known as microhomology-mediated end joining abbreviated as MMEJ. MMEJ is the most mutagenic DSB repair mechanism due to generating deletions, insertion of various sizes, and other genome rearrangements in mammalians. As shown in figure 3, the processes of mtDNA insertion and DSB repair include few steps which are DNA segment alignment, DNA end-processing, DNA synthesis, and ligation. In each step, certain protein complexes are required to facilitate the occurrence of the indicated events. As shown in figure 3, in NHEJ, the Ku70/Ku80 heterodimer and DNA-dependent protein kinase (DNA-PK), for bringing DNA fragments end together, the Artemis nuclease and polynucleotide kinase 3' phosphatase (PNKP) , for the end processing, X family DNA polymerases (Pol μ and Pol λ) and terminal deoxynucleotidyl transferase (TdT) , for DNA synthesis, and XLF/XRCC4/LigIV complex, for completing the repair and joining the ends via a phosphodiester bond, are the protein complexes involved in DSB repair process in many higher organisms. DNA polymerases (Pol μ and Pol λ) and XLF/XRCC4/LigIV complex are shared between two NHEJ and MMEJ repair machinery and have the same responsibility in both repair processes. The first step of MMEJ is done by WRN , Artemis, DNA-PK , and XRCC4 protein complexes which process the ends of DSB and mtDNA fragments in addition to aligning them in order for polymerases and ligases to be able to complete NUMT insertion (figure 3). Post-insertion modification:The complex pattern of NUMT in comparison with the single mitochondrial piece, the appearance of non-continuous mitochondrial DNA in the nuclear genome, and possibly, different orientation of these fragments are the evidence of post-insertion processes of NUMT within the nuclear genome. The causation of these complex patterns might be the result of multiple NUMT insertions at insertional hotspots. In addition, duplication after insertion contributes to NUMT diversity. NUMTs have no self-replicating mechanism or transposition mechanism; therefore, NUMT duplication is expected to occur in tandem or to involve larger segmental duplication at rates representative of the rest of the genome. Evidence for NUMT duplications that are not in proximity to other NUMTs is present in many genomes and probably happens as part of segmental duplication. However, duplications of recent human-specific NUMTs as part of segmental duplication seem to be rare; in humans, only a few NUMTs are found to have overlap with segmental duplication, and those NUMTs were found in only one of the copies while missing from the others, clearly demonstrating that the NUMTs were inserted subsequent to the duplication events. Deletion is another NUMT post-insertional modification method that has not yet been studied in the same amount of detail as an insertion. Constant erosion of phylogenic signals and high mutation rate in animal mtDNA make recognition of such modification, especially deletion, difficult. Studying the cases in which the presence–absence pattern of NUMTs does not agree with the phylogenetic tree, should make detection of recent NUMT losses possible by the means of using multiple genome alignments with the presence of an outgroup. Bensasson and his team members used this method to estimate the oldest inserted NUMT in human, which dated around 58 million years ago.