Newly Discovered Functions of Pseudogenes

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Abstract

Researchers have noted that there are portions of the DNA that look similar to functional genes, but contain lesions or premature stop codons. These genes have been assumed to be largely non-functional, but recent research suggests that many of these ‘pseudogenes’ are actually functional.  This paper is an overview of some of the research done in the area of  pseudogene functionality. I address several recent advances in the  area of genetic research regarding pseudogene functionality  chronologically, starting from one of the first discoveries of a  functional pseudogene and ending with a paper from this year (2013). Broadly speaking, it would seem that the assumption of non-functionality has been overturned regarding many pseudogenes, and the evidence suggests that many more pseudogenes may have a function that has yet to be discovered.

Screen Shot 2015-10-27 at 9.22.09 PMPseudogenes have been typically understood as portions of DNA  that have lost their function and remain in the DNA as a relic that  signifies past functionality. The prefix ‘pseudo-‘ indicates that  something is fake or false, and a pseudogene is a portion of DNA  that looks like a functioning gene, but is not actually functional. Pseudogenes have been placed in the ‘junk DNA’ category, ‘dead’, non-functional by-products of evolution. If a pseudogene is transcribed at all, it is often considered to be largely a neutral process that hasn’t been weeded out by selection. However, recent evidence has shown that many pseudogenes have very important functions in the genome of nearly every organism, humans included. There are very good reasons to revise the definition of ‘pseudogene’ to include a wide variety of biological functions, from gene expression and cellular function to gene regulation and tumor suppression. The newly discovered functions are making the term ‘pseudogene’ notoriously ambiguous. This review will analyze a small handful of functions discovered for pseudogenes that were previously assumed to be non-functional byproducts of genome evolution. It is not intended to be an exhaustive treatment of newly discovered pseudogene functionality. Functions are being ascribed to pseudogenes on a fairly regular basis in contemporary genetics literature, and some of the literature is reviewed in chronological order.

Historically, many biologists have avoided ascribing functions to pseudogenes because they do not directly code for proteins. Since the 1970s, researchers have noticed that portions of the DNA look similar to protein-coding genes but contain lesions or premature stop codons. The term pseudogene was used to signify these ‘lookalikes’.  A gene is obviously functional if it produces a functional protein product, but these pseudogenes have interruptions or mutations that would prevent a functioning protein from forming. The conclusion that these genes are non-functional is a byproduct of the neutral theory of molecular evolution. (Zheng & Gerstein, 2007) In recent times, many of these pseudogenes have been found to perform vital functions, making the term “functionless pseudogene” a misnomer. This has created difficulty in defining exactly what is meant by the term pseudogene, and many have proposed a revision of the current understanding of pseudogenes. (Podlaha & Zhang, 2010) Some have begun replacing the word “pseudogene” with “potogene”, which are “DNA sequences with a potentiality for becoming new genes”. (Balakirev & Ayala, 2003)

A paper written by Dr. Yoshihisa Yano and colleagues in 2004 was an early example of definitive functionality for pseudogenes. In this study, they mutated a mouse’s DNA in a section associated with pseudogene Makorin1-p1. The insertion caused a change in transcription, which resulted in the destabilization of the Makorin-1 mRNA, the mRNA product of the pseudogenes’ homologous coding gene. The result of this induced change was that the mouse had polycystic kidneys, severe bone deformities and also “exhibited failure to thrive”. This discovery was one of the first insights into the important functions of pseudogenes in gene regulation. (Yano et. al, 2004)

Research done by Dr. Deyou Zheng and colleagues in 2005 provides evidence for the transcription of approximately one fifth of the pseudogenes found on human chromosome 22. According to the research, there are 525 pseudogenes on human chromosome 22, and 154 of those are processed. They found that around 12% of the pseudogenes have upstream CpG islands. This is intriguing because, according to Zheng and colleagues, “… CpG islands are usually associated with transcriptional promoters…”. Ultimately, they discovered that pseudogenes are less conserved than protein-coding genes, but they are preserved more than the intergenic background. (Zheng, et al, 2005) This indicates that the protein-coding genes are more important to cellular function, but the pseudogenes are more important than the intergenic background. In 2007, Dr. Deyou Zheng and Dr. Mark B. Gerstein showed that pseudogene transcription has been observed in many places, providing “a conservative estimate that 5-20% of human pseudogenes are transcriptionally active”. (Zheng & Gerstein, 2007)

A study done on small interfering RNAs in mouse oocytes has shown that pseudogenes play a vital function in gene regulation by way of RNA interference pathways. In this study, Dr. Oliver H. Tam and colleagues show that “a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes”. These pseudogene-derived endo-siRNAs perform a wide variety of functions, including the direct generation of small RNAs, the repression of genetic elements and the regulation of the protein, Dicer. Dicer plays an integral role in the production of RNA. (Tam et. al, 2008)

In 2010, Dr. Laura Poliseno and colleagues discovered that pseudogenes play a functional role in RNA competition in cancer cells. Based on the fact that microRNAs bind to RNAs, the RNAs likely possess a biological role (independent of protein-coding functionality) that relies upon their ability to compete for microRNA binding sites.  The researchers made this inference by observing the “functional relationship between the mRNAs produced by the PTEN tumour suppressor gene and its pseudogene (PTENP1) and the critical consequences of this interaction”. The pseudogene PTENP1 plays a regulatory role in the production of PTEN and has a growth-suppression role. The PTENP1 is also selectively lost in human cancer cells, which lends credence to the idea that it is functionally active in cancer. (Poliseno et. al, 2010)regulation by way of RNA interference pathways. In this study, Dr. Oliver H. Tam and colleagues show that “a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes”. These pseudogene-derived endo-siRNAs perform a wide variety of functions, including the direct generation of small RNAs, the repression of genetic elements and the regulation of the protein, Dicer. Dicer plays an integral role in the production of RNA. (Tam et. al, 2008)ne fifth of the pseudogenes found on human chromosome 22. According to the research, there are 525 pseudogenes on human chromosome 22, and 154 of those are processed. They found that around 12% of the pseudogenes have upstream CpG islands. This is intriguing because, according to Zheng and colleagues, “… CpG islands are usually associated with transcriptional promoters…”. Ultimately, they discovered that pseudogenes are less conserved than protein-coding genes, but they are preserved more than the intergenic background. (Zheng, et al, 2005) This indicates that the protein-coding genes are more important to cellular function, but the pseudogenes are more important than the intergenic background. In 2007, Dr. Deyou Zheng and Dr. Mark B. Gerstein showed that pseudogene transcription has been observed in many places, providing “a conservative estimate that 5-20% of human pseudogenes are transcriptionally active”. (Zheng & Gerstein, 2007)

According to a research done by Dr. Grainne McEntee and colleagues, the pseudogene dihydrogolate reductase-like 1 (DHFRL1) is now a “former pseudogene” based on the function discovered for it.  Prior to discovery, the DHFRL1 former-pseudogene was assumed to be unexpressed and non-functional. Their research has shown that this pseudogene actually encodes the enzyme, dihydrofolate reductase. This is the second gene in the human genome discovered with this function, the first being DHFR. (McEntee et. al, 2011)

Research done by Dr. Ryan Charles Pink and colleagues has challenged  both the concept of a pseudogene, as well as the term junk-DNA. They  show that while pseudogenes may not be directly responsible for protein  coding, that does not indicate that it is functionless. They show that  many pseudogenes have regulatory roles in the cell, regulating many aspects of protein transcription, translation and function. Many pseudogenes are transcribed into RNA, have tissue-specific patterns of activation, regulate proteins through the RNAi pathway, contribute to normal cellular regulation and play vital roles in cancer. The “pseudogenes are capable of regulating tumor suppressors and oncogenes by acting as microRNA decoys” and many pseudogenes are deregulated during cancer, which indicates they perform a function associated with cancer regulation. (Pink et. al, 2011)

Dr. Ana Moleirinho and colleagues discovered that human hemoglobin contains 1 alpha-like globin monomer and 2 beta-like globin monomers. Within the beta-like globin monomers, there are five genes and 1 pseudogene. The pseudogene plays an active role in the monomer, and the expression of the gene “undergoes two critical switches: the embryonic-to-fetal and fetal-to-adult transition.” They also show that both “HBD and pseudogene HBBP1 have evolved under purifying selection, suggesting that their roles are essential and nonredundant”. The pseudogene may also play a regulatory role in the ontogenic switches of gene expression. (Moleirinho et. al, 2013)

Collectively, these studies show that many pseudogenes are perform vital tasks and are not non-functional. While not as highly conserved as their protein-coding partners, pseudogenes often show moderate to high levels of conservation. They also play a demonstrable role in gene regulation and expression. This information can inspire further research in an attempt to discover the true function of portions of the DNA often assumed to be non-functional. Using this research to assess the future, we can predict that many more pseudogenes will be discovered to have important roles. This research will help us understand the way genetics influences the function of cells, tissues and entire organisms. By understanding our genome better, we may be able to understand and cure deadly genetic diseases such as cancer.

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References

  • Balakirev, Evgeniy S., and Francisco J. Ayala. 2003. “Pseudogenes: Are They ‘Junk’ or Functional DNA?” Annual Review of Genetics
  • McEntee, Grainne, S. Minguzzi, K. O’Brien, N. Ben Larbi, C. Loscher, C. O’Fagain, A. Parle-McDermott. 2011. “The former annotated human pseudogene dihydrofolate reductase-like 1 (DHFRL1) is expressed and functional” PNAS
  • Moleirinho, Ana, S. Seixas, A. M. Lopes, C. Bento, M. J. Prata, and A. Amorim. 2013. “Evolutionary Constraints in the b-Globin Cluster: The Signature of Purifying Selection at the d-Globin (HBD) Locus and Its Role in Developmental Gene Regulation” Genome Biology and Evolution
  • Pink, Ryan Charles, Kate Wicks, Daniel Paul Caley, et al. 2011. “Pseudogenes: Pseudo-functional or key regulators in health and disease?” RNA: A Publication of the RNA Society
  • Podlaha, Ondrej & Jianzhi Zhang. 2010. “Pseudogenes and Their Evolution”. Encyclopedia of Life Sciences (ELS)
  • Poliseno, Laura, L. Salmena, J. Shang, B. Carver, W. J. Haveman, and P. P. Pandolfi. 2010. “A coding-dependent function of gene and pseudogene mRNAs regulates tumor biology”. Nature.
  • Tam, Oliver H., A. A. Aravin, P. Stein, A. Girard, E. P. Murchison, S. Cheloufi, E. Hodges, M. Anger, R. Sachidanandam, R. M. Schultz, G. J. Hannon. 2008. “Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes”. Nature.
  • Yano, Yoshihisa, R. Saito, N. Yoshida, A. Toshiki, A. Wynshaw-Boris, M. Tomita, S. Hirotsune. 2004. “A new role for expressed pseudogenes as ncRNA: regulation of mRNA stability of its homologous coding gene”. Journal of Molecular Medicine.
  • Zheng, Deyou. Z. Zhang, P. M. Harrison, J. Karro, N. Carriero and M. Gerstein. 2005. “Integrated Pseudogene Annotation for Human Chromosome 22: Evidence for Transcription”. Journal of Molecular Biology
  • Zheng, Deyou & M. B. Gerstein. 2007. The ambiguous boundary between genes and pseudogenes: the dead rise up, or do they?” Trends in Genetics.
  • Zheng, Deyou & M. B. Gerstein. 2007. The ambiguous boundary between genes and pseudogenes: the dead rise up, or do they?” Trends in Genetics.

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This was a research project done in Science Writing (BIO389W) during the Fall 2013 semester.

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