Non-coding DNA sequences are components of an organism's DNA that do not encode protein sequences. Some non-coding DNA is transcribed into functional non-coding RNA molecules (e.g. transfer RNA, ribosomal RNA, and regulatory RNAs). Other functions of non-coding DNA include the transcriptional and translational regulation of protein-coding sequences, scaffold attachment regions, origins of DNA replication, centromeres and telomeres. Its RNA counterpart is non-coding RNA.
The amount of non-coding DNA varies greatly among species. Often, only a small percentage of the genome is responsible for coding proteins, but an increasing percentage is being shown to have regulatory functions. When there is much non-coding DNA, a large proportion appears to have no biological function, as predicted in the 1960s. Since that time, this non-functional portion has controversially been called "junk DNA".
The international Encyclopedia of DNA Elements (ENCODE) project uncovered, by direct biochemical approaches, that at least 80% of human genomic DNA has biochemical activity such as "transcription, transcription factor association, chromatin structure, and histone modification". Though this was not necessarily unexpected due to previous decades of research discovering many functional non-coding regions, some scientists criticized the conclusion for conflating biochemical activity with biological function. Estimates for the biologically functional fraction of the human genome based on comparative genomics range between 8 and 15%. However, others have argued against relying solely on estimates from comparative genomics due to its limited scope since non-coding DNA has been found to be involved in epigenetic activity and complex networks of genetic interactions and is explored in evolutionary developmental biology.
The amount of total genomic DNA varies widely between organisms, and the proportion of coding and non-coding DNA within these genomes varies greatly as well. For example, it was originally suggested that over 98% of the human genome does not encode protein sequences, including most sequences within introns and most intergenic DNA, while 20% of a typical prokaryote genome is non-coding.
In eukaryotes, genome size, and by extension the amount of non-coding DNA, is not correlated to organism complexity, an observation known as the C-value enigma. For example, the genome of the unicellular Polychaos dubium (formerly known as Amoeba dubia) has been reported to contain more than 200 times the amount of DNA in humans. The pufferfish Takifugu rubripes genome is only about one eighth the size of the human genome, yet seems to have a comparable number of genes; approximately 90% of the Takifugu genome is non-coding DNA. Therefore, most of the difference in genome size is not due to variation in amount of coding DNA, rather, it is due to a difference in the amount of non-coding DNA.
In 2013, a new "record" for the most efficient eukaryotic genome was discovered with Utricularia gibba, a bladderwort plant that has only 3% non-coding DNA and 97% of coding DNA. Parts of the non-coding DNA were being deleted by the plant and this suggested that non-coding DNA may not be as critical for plants, even though non-coding DNA is useful for humans. Other studies on plants have discovered crucial functions in portions of non-coding DNA that were previously thought to be negligible and have added a new layer to the understanding of gene regulation.
Main article: Conserved non-coding sequence
Cis-regulatory elements are sequences that control the transcription of a nearby gene. Many such elements are involved in the evolution and control of development. Cis-elements may be located in 5' or 3' untranslated regions or within introns. Trans-regulatory elements control the transcription of a distant gene.
Promoters facilitate the transcription of a particular gene and are typically upstream of the coding region. Enhancer sequences may also exert very distant effects on the transcription levels of genes.
Introns are non-coding sections of a gene, transcribed into the precursor mRNA sequence, but ultimately removed by RNA splicing during the processing to mature messenger RNA. Many introns appear to be mobile genetic elements.
Studies of group I introns from Tetrahymena protozoans indicate that some introns appear to be selfish genetic elements, neutral to the host because they remove themselves from flanking exons during RNA processing and do not produce an expression bias between alleles with and without the intron. Some introns appear to have significant biological function, possibly through ribozyme functionality that may regulate tRNA and rRNA activity as well as protein-coding gene expression, evident in hosts that have become dependent on such introns over long periods of time; for example, the trnL-intron is found in all green plants and appears to have been vertically inherited for several billions of years, including more than a billion years within chloroplasts and an additional 2–3 billion years prior in the cyanobacterial ancestors of chloroplasts.
Pseudogenes are DNA sequences, related to known genes, that have lost their protein-coding ability or are otherwise no longer expressed in the cell. Pseudogenes arise from retrotransposition or genomic duplication of functional genes, and become "genomic fossils" that are nonfunctional due to mutations that prevent the transcription of the gene, such as within the gene promoter region, or fatally alter the translation of the gene, such as premature stop codons or frameshifts. Pseudogenes resulting from the retrotransposition of an RNA intermediate are known as processed pseudogenes; pseudogenes that arise from the genomic remains of duplicated genes or residues of inactivated genes are nonprocessed pseudogenes. Transpositions of once functional mitochondrial genes from the cytoplasm to the nucleus, also known as NUMTs, also qualify as one type of common pseudogene. Numts occur in many eukaryotic taxa.
While Dollo's Law suggests that the loss of function in pseudogenes is likely permanent, silenced genes may actually retain function for several million years and can be "reactivated" into protein-coding sequences and a substantial number of pseudogenes are actively transcribed. Because pseudogenes are presumed to change without evolutionary constraint, they can serve as a useful model of the type and frequencies of various spontaneous genetic mutations.
Transposons and retrotransposons are mobile genetic elements. Retrotransposon repeated sequences, which include long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), account for a large proportion of the genomic sequences in many species. Alu sequences, classified as a short interspersed nuclear element, are the most abundant mobile elements in the human genome. Some examples have been found of SINEs exerting transcriptional control of some protein-encoding genes.
Endogenous retrovirus sequences are the product of reverse transcription of retrovirus genomes into the genomes of germ cells. Mutation within these retro-transcribed sequences can inactivate the viral genome.
Over 8% of the human genome is made up of (mostly decayed) endogenous retrovirus sequences, as part of the over 42% fraction that is recognizably derived of retrotransposons, while another 3% can be identified to be the remains of DNA transposons. Much of the remaining half of the genome that is currently without an explained origin is expected to have found its origin in transposable elements that were active so long ago (> 200 million years) that random mutations have rendered them unrecognizable. Genome size variation in at least two kinds of plants is mostly the result of retrotransposon sequences.
Telomeres are regions of repetitive DNA at the end of a chromosome, which provide protection from chromosomal deterioration during DNA replication. Recent studies have shown that telomeres function to aid in its own stability. Telomeric repeat-containing RNA (TERRA) are transcripts derived from telomeres. TERRA has been shown to maintain telomerase activity and lengthen the ends of chromosomes.
The term "junk DNA" became popular in the 1960s. According to T. Ryan Gregory, the nature of junk DNA was first discussed explicitly in 1972 by a genomic biologist, David Comings, who applied the term to all non-coding DNA. The term was formalized that same year by Susumu Ohno, who noted that the mutational load from deleterious mutations placed an upper limit on the number of functional loci that could be expected given a typical mutation rate. Ohno hypothesized that mammal genomes could not have more than 30,000 loci under selection before the "cost" from the mutational load would cause an inescapable decline in fitness, and eventually extinction. This prediction remains robust, with the human genome containing approximately (protein-coding) 20,000 genes. Another source for Ohno's theory was the observation that even closely related species can have widely (orders-of-magnitude) different genome sizes, which had been dubbed the C-value paradox in 1971.
The term "junk DNA" has been questioned on the grounds that it provokes a strong a priori assumption of total non-functionality and some have recommended using more neutral terminology such as "non-coding DNA" instead. Yet "junk DNA" remains a label for the portions of a genome sequence for which no discernible function has been identified and that through comparative genomics analysis appear under no functional constraint suggesting that the sequence itself has provided no adaptive advantage.
Since the late 70s it has become apparent that the majority of non-coding DNA in large genomes finds its origin in the selfish amplification of transposable elements, of which W. Ford Doolittle and Carmen Sapienza in 1980 wrote in the journal Nature: "When a given DNA, or class of DNAs, of unproven phenotypic function can be shown to have evolved a strategy (such as transposition) which ensures its genomic survival, then no other explanation for its existence is necessary." The amount of junk DNA can be expected to depend on the rate of amplification of these elements and the rate at which non-functional DNA is lost. In the same issue of Nature, Leslie Orgel and Francis Crick wrote that junk DNA has "little specificity and conveys little or no selective advantage to the organism". The term occurs mainly in popular science and in a colloquial way in scientific publications, and it has been suggested that its connotations may have delayed interest in the biological functions of non-coding DNA.
Some evidence indicate that some "junk DNA" sequences are sources for (future) functional activity in evolution through exaptation of originally selfish or non-functional DNA.
In 2012, the ENCODE project, a research program supported by the National Human Genome Research Institute, reported that 76% of the human genome's non-coding DNA sequences were transcribed and that nearly half of the genome was in some way accessible to genetic regulatory proteins such as transcription factors. However, the suggestion by ENCODE that over 80% of the human genome is biochemically functional has been criticized by other scientists, who argue that neither accessibility of segments of the genome to transcription factors nor their transcription guarantees that those segments have biochemical function and that their transcription is selectively advantageous. After all, non-functional sections of the genome can be transcribed, given that transcription factors typically bind to short sequences that are found (randomly) all over the whole genome.
Furthermore, the much lower estimates of functionality prior to ENCODE were based on genomic conservation estimates across mammalian lineages. Widespread transcription and splicing in the human genome has been discussed as another indicator of genetic function in addition to genomic conservation which may miss poorly conserved functional sequences. Furthermore, much of the apparent junk DNA is involved in epigenetic regulation and appears to be necessary for the development of complex organisms. Genetic approaches may miss functional elements that do not manifest physically on the organism, evolutionary approaches have difficulties using accurate multispecies sequence alignments since genomes of even closely related species vary considerably, and with biochemical approaches, though having high reproducibility, the biochemical signatures do not always automatically signify a function. Kellis et al. noted that 70% of the transcription coverage was less than 1 transcript per cell (and may thus be based on spurious background transcription). On the other hand, they argued that 12–15% fraction of human DNA may be under functional constraint, and may still be an underestimate when lineage-specific constraints are included. Ultimately genetic, evolutionary, and biochemical approaches can all be used in a complementary way to identify regions that may be functional in human biology and disease. Some critics have argued that functionality can only be assessed in reference to an appropriate null hypothesis. In this case, the null hypothesis would be that these parts of the genome are non-functional and have properties, be it on the basis of conservation or biochemical activity, that would be expected of such regions based on our general understanding of molecular evolution and biochemistry. According to these critics, until a region in question has been shown to have additional features, beyond what is expected of the null hypothesis, it should provisionally be labelled as non-functional.
Some non-coding DNA sequences must have some important biological function. This is indicated by comparative genomics studies that report highly conserved regions of non-coding DNA, sometimes on time-scales of hundreds of millions of years. This implies that these non-coding regions are under strong evolutionary pressure and positive selection. For example, in the genomes of humans and mice, which diverged from a common ancestor 65–75 million years ago, protein-coding DNA sequences account for only about 20% of conserved DNA, with the remaining 80% of conserved DNA represented in non-coding regions. Linkage mapping often identifies chromosomal regions associated with a disease with no evidence of functional coding variants of genes within the region, suggesting that disease-causing genetic variants lie in the non-coding DNA. The significance of non-coding DNA mutations in cancer was explored in April 2013.
Non-coding genetic polymorphisms play a role in infectious disease susceptibility, such as hepatitis C. Moreover, non-coding genetic polymorphisms contribute to susceptibility to Ewing sarcoma, an aggressive pediatric bone cancer.
Some specific sequences of non-coding DNA may be features essential to chromosome structure, centromere function and recognition of homologous chromosomes during meiosis.
According to a comparative study of over 300 prokaryotic and over 30 eukaryotic genomes, eukaryotes appear to require a minimum amount of non-coding DNA. The amount can be predicted using a growth model for regulatory genetic networks, implying that it is required for regulatory purposes. In humans the predicted minimum is about 5% of the total genome.
Over 10% of 32 mammalian genomes may function through the formation of specific RNA secondary structures. The study used comparative genomics to identify compensatory DNA mutations that maintain RNA base-pairings, a distinctive feature of RNA molecules. Over 80% of the genomic regions presenting evolutionary evidence of RNA structure conservation do not present strong DNA sequence conservation.
Non-coding DNA may perhaps serve to decrease the probability of gene disruption during chromosomal crossover.
Genome-wide association studies (GWAS) and machine learning analysis of large genomic datasets has led to the construction of polygenic predictors for human traits such as height, bone density, and many disease risks. Similar predictors exist for plant and animal species and are used in agricultural breeding. The detailed genetic architecture of human predictors has been analyzed and significant effects used in prediction are associated with DNA regions far outside coding regions. The fraction of variance accounted for (i.e., fraction of predictive power captured by the predictor) in coding vs. non-coding regions varies widely for different complex traits. For example, atrial fibrillation and coronary artery disease risk are mostly controlled by variants in non-coding regions (non-coding variance fraction over 70 percent), whereas diabetes and high cholesterol display the opposite pattern (non-coding variance roughly 20-30 percent). Individual differences between humans are clearly affected in a significant way by non-coding genetic loci, which is strong evidence for functional effects. Whole exome genotypes (i.e., which contain information restricted to coding regions only) do not contain enough information to build or even evaluate polygenic predictors for many well-studied complex traits and disease risks.
In 2013, it was estimated that, in general, up to 85% of GWAS loci have non-coding variants as the likely causal association. The variants are often common in populations and were predicted to affect disease risks through small phenotypic effects, as opposed to the large effects of Mendelian variants.
Main article: Regulation of gene expression
Some non-coding DNA sequences determine the expression levels of various genes, both those that are transcribed to proteins and those that themselves are involved in gene regulation.
Main article: Transcription factor
Some non-coding DNA sequences determine where transcription factors attach. A transcription factor is a protein that binds to specific non-coding DNA sequences, thereby controlling the flow (or transcription) of genetic information from DNA to mRNA.
Main article: Operon § Operator
An operator is a segment of DNA to which a repressor binds. A repressor is a DNA-binding protein that regulates the expression of one or more genes by binding to the operator and blocking the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes. This blocking of expression is called repression.
Main article: Enhancer (genetics)
An enhancer is a short region of DNA that can be bound with proteins (trans-acting factors), much like a set of transcription factors, to enhance transcription levels of genes in a gene cluster.
Main article: Silencer (genetics)
A silencer is a region of DNA that inactivates gene expression when bound by a regulatory protein. It functions in a very similar way as enhancers, only differing in the inactivation of genes.
Main article: Promoter (genetics)
A promoter is a region of DNA that facilitates transcription of a particular gene when a transcription factor binds to it. Promoters are typically located near the genes they regulate and upstream of them.
Main article: Insulator (genetics)
A genetic insulator is a boundary element that plays two distinct roles in gene expression, either as an enhancer-blocking code, or rarely as a barrier against condensed chromatin. An insulator in a DNA sequence is comparable to a linguistic word divider such as a comma in a sentence, because the insulator indicates where an enhanced or repressed sequence ends.
Shared sequences of apparently non-functional DNA are a major line of evidence of common descent.
Pseudogene sequences appear to accumulate mutations more rapidly than coding sequences due to a loss of selective pressure. This allows for the creation of mutant alleles that incorporate new functions that may be favored by natural selection; thus, pseudogenes can serve as raw material for evolution and can be considered "protogenes".
A study published in 2019 shows that new genes (termed de novo gene birth) can be fashioned from non-coding regions. Some studies suggest at least one-tenth of genes could be made in this way.
A statistical distinction between coding and non-coding DNA sequences has been found. It has been observed that nucleotides in non-coding DNA sequences display long range power law correlations while coding sequences do not.
Police sometimes gather DNA as evidence for purposes of forensic identification. As described in Maryland v. King, a 2013 U.S. Supreme Court decision:
The current standard for forensic DNA testing relies on an analysis of the chromosomes located within the nucleus of all human cells. 'The DNA material in chromosomes is composed of "coding" and "non-coding" regions. The coding regions are known as genes and contain the information necessary for a cell to make proteins. . . . Non-protein coding regions . . . are not related directly to making proteins, [and] have been referred to as "junk" DNA.' The adjective "junk" may mislead the lay person, for in fact this is the DNA region used with near certainty to identify a person.
Comings (1972), on the other hand, gave what must be considered the first explicit discussion of the nature of "junk DNA," and was the first to apply the term to all non-coding DNA."; "For this reason, it is unlikely that any one function for non-coding DNA can account for either its sheer mass or its unequal distribution among taxa. However, dismissing it as no more than "junk" in the pejorative sense of "useless" or "wasteful" does little to advance the understanding of genome evolution. For this reason, the far less loaded term "noncoding DNA" is used throughout this chapter and is recommended in preference to "junk DNA" for future treatments of the subject."
The term "junk DNA" repelled mainstream researchers from studying noncoding genetic material for many years