Because pseudogenes are generally thought of as the last stop for genomic material that is to be removed from the genome, 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 close to 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.
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. Other researchers have since hypothesized similar roles for other pseudogenes. A bioinformatics analysis has shown that processed pseudogenes can be inserted into introns of annotated genes and be incorporated into alternatively spliced transcripts. 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 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 phosphoglycerate mutase 3 () actually produces a functional protein.
Two 2008 publications in Nature discuss that some endogenous siRNAs are derived from pseudogenes, and thus some pseudogenes play a role in regulating protein-coding transcripts. In June 2010, Nature published an article showing the mRNA levels of tumour suppressor PTEN and oncogenic KRAS is affected by their pseudogenes PTENP1 and KRAS1P. This discovery demonstrated an miRNA decoy function for pseudogenes and identified their transcripts as biologically active units in tumor biology; thus attributing a novel biological role to expressed pseudogenes, as they can regulate coding gene expression, and reveal a non-coding function for mRNAs in disease progression.
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