Pseudogenes are defunct relatives of known genes that have lost their protein-coding ability or are otherwise no longer expressed in the cell. Although they may have some gene-like features (such as Promoters, CpG islands, and splice sites), they are nonetheless considered nonfunctional, due to their lack of protein-coding ability resulting from various genetic disablements (stop codons, frameshifts, or a lack of transcription) or their inability to function as an RNA (such as with rRNA pseudogenes). Thus the term, coined in 1977 by Jacq, et al., is composed of the prefix pseudo, which means false, and the root gene, which is the central unit of molecular genetics.
Because pseudogenes are generally thought of the last stop for genomic material that is to be removed from the genome, they are often labeled as junk DNA. Nonetheless, pseudogenes contain fascinating biological and evolutionary histories within their sequences. This is due to a pseudogene's shared ancestry with a functional gene: in the same way that Darwin thought of two species as possibly having a shared common ancestry followed by millions of years of evolutionary divergence (see speciation), a pseudogene and its associated functional gene also share a common ancestor and have diverged as separate genetic entities over millions of years.
Pseudogenes are characterized by a combination of homology to a known gene and nonfunctionality. That is, although every pseudogene has a DNA sequence that is similar to some functional gene, they are nonetheless unable to produce functional final products (nonfunctionality).
Pseudogenes are quite difficult to identify and characterize in genomes, because the two requirements of homology and nonfunctionality are implied through sequence calculations and alignments rather than biologically proven.
Homology is implied by sequence identity between the DNA sequences of the pseudogene and parent gene. After aligning the two sequences, the percentage of identical base pairs is computed. A high sequence identity (usually between 40% and 100%) means that it is highly likely that these two sequences diverged from a common ancestral sequence (are homologous), and highly unlikely that these two sequences were independently created (see typewriting monkeys).
Nonfunctionality can manifest itself in many ways. Normally, a gene must go through several steps in going from a genetic DNA sequence to a fully-functional protein: transcription, pre-mRNA processing, translation, and protein folding are all required parts of this process. If any of these steps fails, then the sequence may be considered nonfunctional. In high-throughput pseudogene identification, the most commonly identified disablements are stop codons and frameshifts, which almost universally prevent the translation of a functional protein product.
Pseudogenes for RNA genes are often easier to discover. Many RNA genes occur as multiple copy genes, and pseudogenes are identified through sequence identity and location within the region.
Types and origin of pseudogenes
There are three main types of pseudogenes, all with distinct mechanisms of origin and characteristic features. The classifications of pseudogenes are as follows:
Processed (or retrotransposed) pseudogenes. In higher eukaryotes, particularly mammals, retrotransposition is a fairly common event that has had a huge impact on the composition of the genome. For example, somewhere between 30% - 44% of the human genome consists of repetitive elements such as SINEs and LINEs (see retrotransposons). In the process of retrotransposition, a portion of the mRNA transcript of a gene is spontaneously reverse transcribed back into DNA and inserted into chromosomal DNA. Although retrotransposons usually create copies of themselves, it has been shown in an in vitro system that they can create retrotransposed copies of random genes, too. Once these pseudogenes are inserted back into the genome, they usually contain spliced-out introns and a Poly-A tail, two hallmarks features of cDNAs. However, because they are derived from a mature mRNA product, processed pseudogenes also lack the upstream promoters of normal genes; thus, they are considered "dead on arrival", becoming non-functional pseudogenes immediately upon the retrotransposition event. A further characteristic of processed pseudogenes is common truncation of the 5' end relative to the parent sequence, which is a result of the relatively non-processive retrotransposition mechanism that creates processed pseudogenes.
Non-processed (or duplicated) pseudogenes. Gene duplication is another common and important process in the evolution of genomes. A copy of a functional gene may arise as a result of a gene duplication event and subsequently acquire mutations that cause it to become nonfunctional. Duplicated pseudogenes usually have all the same characteristics of genes, including an intact exon-intron structure and promoter sequences. The loss of a duplicated gene's functionality usually has little effect on an organism's fitness, since an intact functional copy still exists. According to some evolutionary models, shared duplicated pseudogenes indicate the evolutionary relatedness of humans and the other primates.
Disabled genes, or unitary pseudogenes. Various mutations can stop a gene from being successfully transcribed or translated, and a gene may become nonfunctional or deactivated if such a mutation becomes fixed in the population. This is the same mechanism by which non-processed genes become deactivated, but the difference in this case is that the gene was not duplicated before becoming disabled. Normally, such gene deactivation would be unlikely to become fixed in a population, but various population effects, such as genetic drift, a population bottleneck, or in some cases, natural selection, can lead to fixation. The classic example of a unitary pseudogene is the gene that presumably coded the enzyme L-gulono-γ-lactone oxidase (GLO) in primates. In all mammals besides primates (except guinea pigs), GLO aids in the biosynthesis of Ascorbic acid (vitamin C), but it exists as a disabled gene in humans and other primates. Another interesting and more recent example of a disabled gene, which links the deactivation of a caspase gene (through a nonsense mutation) to positive selection in humans, can be found in Xue et al. 2006.
Pseudogenes can complicate molecular genetic studies. For example, a researcher who wants to amplify a gene by PCR may simultaneously amplify a pseudogene that shares similar sequences. This is known as PCR bias or amplification bias. Similarly, pseudogenes are sometimes annotated as genes in genome sequences.
Processed pseudogenes often pose a problem for gene prediction programs, often being misidentified as real genes or exons. It has been proposed that identification of processed pseudogenes can help improve the accuracy of gene prediction methods.
It has also been shown that the parent sequences that give rise to processed pseudogenes lose their coding potential faster than those giving rise to non-processed pseudogenes.
It has been established that quite a few pseudogenes can go through the process of transcription, either if their own promoter is still intact or in some cases using the promoter of a nearby gene; this expression of pseudogenes also appears to be tissue-specific. In 2003, Hirotsune et al. identified a retrotransposed pseudogene whose transcript purportedly plays a trans-regulatory role in the expression of its homologous gene, Makorin1, and suggested this as a general model under which pseudogenes may play an important biological role. Other researchers have since hypothesized similar roles for other pseudogenes. Hirotsune's report prompted two molecular biologists to carefully review scientific literature on the subject of pseudogenes. To the surprise of many, they found a number of examples in which pseudogenes play a role in gene regulation and expression, forcing Hirotsune's group to rescind their claim that they were the first to identify pseudogene function. Furthermore, the original findings of Hirotsune et al. concerning Makorin1 have recently been strongly contested; thus, the tantalizing possibility that some pseudogenes could have important biological functions was disputed.
Additionally, University of Chicago and University of Cincinnati scientists reported in 2002 that a processed pseudogene called Phospholycerate mutase or PGAM3 actually produces a functional protein
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