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Wednesday, November 18, 2015

Different kinds of pseudogenes - are they really pseudogenes?

I define a gene as "DNA sequence that is transcribed to produce a functional product" [What Is a Gene? ]. Genes can encode proteins or the final product can be a functional RNA other than mRNA.

A pseudogene is a broken gene that cannot produce a functional RNA. They are called "pseudogenes" because they resemble active genes but carry mutations that have rendered them nonfunctional. The human genome contains about 14,000 pseudogenes related to protein-coding genes according to the latest Ensembl Genome Reference Consortium Human Genome build [GRCh38.p3]. There's some controversy over the exact number but it's certainly in that ballpark.1

The GENCODE Pseudogene Resource is the annotated database used by Ensembl and ENCODE (Pei et al. 2012).

There are an unknown number of pseudogenes derived from genes for noncoding functional RNAs. These pseudogenes are more difficult to recognize but some of them are present in huge numbers of copies. The Alu elements in the human genome are derived from 7SL RNA and there are similar elements in the mouse genome that are derived from tRNA genes.

There are three main classes of pseudogenes and one important subclass. The categories apply to pseudogenes derived from protein-coding genes and to those derived from genes that specify functional noncoding RNAs. I'm going to describe each of the categories in separate posts. I'll mostly describe them using a protein-coding gene as the parent.

1. Processed pseudogenes [Processed pseudogenes ]
2. Duplicated pseudogenes [Duplicated pseudogenes ]
3. Unitary pseudogenes [Unitary Pseudogenes]
4. subclass: Polymorphic pseudogenes [Polymorphic Pseudogenes]

There are many scientists who think that pseudogenes aren't pseudogenes at all—they think pseudogenes are actually genes with a function that's different from the parent gene. These scientists are not kooks,2 in many cases they are very good scientists. Some of them, like Mark Gerstein and his colleagues, merely raise doubts about whether pseudogenes are junk DNA.
Pseudogenes have long been considered as nonfunctional genomic sequences. However, recent evidence suggests that many of them might have some form of biological activity, and the possibility of functionality has increased interest in their accurate annotation and integration with functional genomic data.
                                                                  Pei et al. (2012)
Other scientists are writing scientific papers that are much more sensationalist ...
Abstract: A paradigm shift is sweeping modern day molecular biology following the realisation that large amounts of “junk” DNA”, thought initially to be evolutionary remnants, may actually be functional. Several recent studies support a functional role for pseudogene-expressed non-coding RNAs in regulating their protein-coding counterparts. Several hundreds of pseudogenes have been reported as transcribed into RNA in a large variety of tissues and tumours. Most studies have focused on pseudogenes expressed in the sense direction, but some reports suggest that pseudogenes can also be transcribed as antisense RNAs (asRNAs). A few examples of key regulatory genes, such as PTEN and OCT4, have in fact been reported to be under the regulation of pseudogene-expressed asRNAs. Here, we review what are known about pseudogene expressed non-coding RNA mediated gene regulation and their roles in the control of epigenetic states.
                                                                  Groen et al. (2014)
Abstract: Pseudogenes have long been labeled as “junk” DNA, failed copies of genes that arise during the evolution of genomes. However, recent results are challenging this moniker; indeed, some pseudogenes appear to harbor the potential to regulate their protein-coding cousins. Far from being silent relics, many pseudogenes are transcribed into RNA, some exhibiting a tissue-specific pattern of activation. Pseudogene transcripts can be processed into short interfering RNAs that regulate coding genes through the RNAi pathway. In another remarkable discovery, it has been shown that pseudogenes are capable of regulating tumor suppressors and oncogenes by acting as microRNA decoys. The finding that pseudogenes are often deregulated during cancer progression warrants further investigation into the true extent of pseudogene function. In this review, we describe the ways in which pseudogenes exert their effect on coding genes and explore the role of pseudogenes in the increasingly complex web of noncoding RNA that contributes to normal cellular regulation.
                                                                 Pink et al. (2011)
There are even some well-respected evolutionary biogists who question the functionality of "pseudogenes."
Pseudogenes: Are They “Junk” or Functional DNA?

Pseudogenes have been defined as nonfunctional sequences of genomic DNA originally derived from functional genes. It is therefore assumed that all pseudogene mutations are selectively neutral and have equal probability to become fixed in the population. Rather, pseudogenes that have been suitably investigated often exhibit functional roles, such as gene expression, gene regulation, generation of genetic (antibody, antigenic, and other) diversity. Pseudogenes are involved in gene conversion or recombination with functional genes. Pseudogenes exhibit evolutionary conservation of gene sequence, reduced nucleotide variability, excess synonymous over nonsynonymous nucleotide polymorphism, and other features that are expected in genes or DNA sequences that have functional roles. We first review the Drosophila literature and then extend the discussion to the various functional features identified in the pseudogenes of other organisms. A pseudogene that has arisen by duplication or retroposition may, at first, not be subject to natural selection if the source gene remains functional. Mutant alleles that incorporate new functions may, nevertheless, be favored by natural selection and will have enhanced probability of becoming fixed in the population. We agree with the proposal that pseudogenes be considered as potogenes, i.e., DNA sequences with a potentiality for becoming new genes.

                                                                 Balakirev and Ayala (2003)

Pseudogenes are not pseudo any more

Recent significant progress toward understanding the function of pseudogenes in protozoa (Trypanosoma brucei), metazoa (mouse) and plants, make it pertinent to provide a brief overview on what has been learned about this fascinating subject. We discuss the regulatory mechanisms of pseudogenes at the post-transcriptional level and advance new ideas toward understanding the evolution of these, sometimes called “garbage genes” or “junk DNA,” seeking to stimulate the interest of scientists and additional research on the subject. We hope this point-of-view can be helpful to scientists working or seeking to work on these and related issues.
                                                                 Wen et al. (2012)
Given this very public controversy in the scientic literature, it should come as no suprise that the popular press has turned this into a big deal by questioning junk DNA. It should also come as no suprise that anti-science writers gleefully report this "paradigm shift" as evidence for creationism.

The truth is that the vast majority of pseudogenes are, in fact, pseudogenes. They are junk DNA. A very small number have secondarily acquired a new function. This has been known for decades and the classice examples are in all the textbooks.

Just because a few pseudogenes in different species have a function does not mean that most of them do.

Let's learn about pseudogenes so we can be better informed about this controversy.

1. Pseudogenes will gradually accumulate mutations so that after millions of years ancient pseudogenes can no longer be recognized as pseudogenes. Different computer algorithms have different cut-offs when determining whether something is a pseudogene or not. This is not the only problem.

2. At least not all of them are kooks. Some of them are kooks.

Balakirev, E.S., and Ayala, F.J. (2003) DNA polymorphism in the β-esterase gene cluster of Drosophila melanogaster. Genetics, 164(2), 533-544. [doi: 10.1146/annurev.genet.37.040103.103949]

Groen, J.N., Capraro, D., and Morris, K.V. (2014) The emerging role of pseudogene expressed non-coding RNAs in cellular functions. The international journal of biochemistry & cell biology, 54:350-355. [doi:10.1016/j.biocel.2014.05.008]

Pei, B., Sisu, C., Frankish, A., Howald, C., Habegger, L., Mu, X.J., Harte, R., Balasubramanian, S., Tanzer, A., and Diekhans, M. (2012) The GENCODE pseudogene resource. Genome Biol. 13:R51. [doi: 10.1186/gb-2012-13-9-r51]

Pink, R.C., Wicks, K., Caley, D.P., Punch, E.K., Jacobs, L., and Carter, D.R.F. (2011) Pseudogenes: pseudo-functional or key regulators in health and disease? RNA, 17:792-798. [doi: 10.1261/rna.2658311]

Wen, Y.-Z., Zheng, L.L., Qu, L.-H., Ayala, F.J., and Lun, Z.-R. (2012) Pseudogenes are not pseudo any more. RNA biology 9:27-32. [doi: 10.4161/rna.9.1.18277]


anonymous said...

Now this particular blog is interesting! I do have one question regarding the definition of "gene" before continuing with "pseudogene":

When inferring gene orthology across lineages, should nontranscribed "regulatory regions" be considered part of “the gene” being compared? Of course, you recognize where I am heading with this, given current practice to that question already affirms the affirmative answer. And no; I am no champion of ENCODE.

I am hoping for a civil exchange, this time.

Piotr Gąsiorowski said...

There are even some well-respected evolutionary biogists who question the functionality of "pseudogenes."

Insist on their functionality, right?

Thanks for this new series. Very interesting!

Larry Moran said...

If you had followed the link to my post on " What Is a Gene" you would have seen that I discussed the point about regulatory sequences. Which of my statements do you disagree with and why?

John Harshman said...

"Pseudogenes exhibit evolutionary conservation of gene sequence, reduced nucleotide variability, excess synonymous over nonsynonymous nucleotide polymorphism, and other features that are expected in genes or DNA sequences that have functional roles."

Is this true? If so, how general is it?

Larry Moran said...

If "true" and "not true" are the only choices then it's not true. There may be a few gene-like sequences that aren't really pseudogenes because they have acquired a function but they seem to be rare.

John Harshman said...

What does that paper use to support this claim?

Jass said...

I find evolution according to the modern evolutionary theory by knowledgeable scientists approved by Larry Moran quite interesting and entertaining.

It (mindless process) "decided "to preserve 90% of useless genome in one species, and almost none in another. Most knowledgeable scientist agree on something but no evidence is yet available.

Joe Felsenstein said...

Quite early on in the pseudogene literature people developed methods for confirming that a gene was a nonexpressed pseudogene, based on rates of substitution at 1st, 2nd, and 3rd codon positions becoming the same.

Here is a more recent paper on some of these issues:

Larry Moran said...

Speaking of mindless .... if you don't have anything to contribute other than whining about your lack of knowledge then don't say anything at all.

Larry Moran said...

BTW, are you prepared to identify yourself and make a bet?

John Harshman said...

So now Joe appears to be saying that the claim in that 2003 paper is generally false. Since it's paywalled I can't check.

anonymous said...

@ Professor Moran

“Which of my statements do you disagree with and why?”

No disagreement really. You are citing a quick and fast catechism that is still current. I just find current orthodoxy often inconsistent; as when the inference of gene orthology across lineages, nontranscribed "regulatory regions" are often considered part of “the gene” being compared.

The notion of gene has been defined and redefined quite a bit since William Bateson and Wilhelm Johannsen first coined the phrase. Perhaps this particular item of vocabulary has outlived its purpose and deserves to be jettisoned given archaic notions can cause undergraduate students much confusion. For example, “How are some alleles dominant and others recessive? What are the molecular mechanisms?” That question comes up every year, and answering can be problematic given “dominance” can be either “cis-dominant” or “trans-dominant”. Both are unquestionably heritable i.e.“genetic” phenomena. However, the latter is clearly the result of a “gene product” as you would define it, but the former really isn’t.

If every gene is ultimately pleiotropic and if every trait is multigenic, then really what is “a” gene, especially when “a” given phenotype may be the result of a great many interactions from a variety of “loci”. I am intrigued with Keller’s and Harel’s suggestion of “dene”, connoting any DNA sequence that plays a role in the cell. Alright, passingly intrigued, I won’t hold my breath.

I found this paper intriguing:

“Thus, in a modern view of the gene and its function it is much more open and complex. It does not longer exist one true and general description;[sic] instead it takes different meaning for different scientists.”

No big deal, I think we agree more than we disagree. I just wanted to clarify and realize that I may have done just the contrary. I concede that such considerations perhaps constitute an unnecessary distraction from this interesting blog. I will retreat back into lurk-mode.

Anonymous said...

People annotating genes in eukaryotes tend to consider regulatory regions as part of the gene. That must make it quite difficult when dealing with less characterized genes. People annotating microbes don't consider regulatory regions as part of the genes.

But orthology is not inferred based on whether the gene includes regulatory regions or not. Orthology is most often inferred from protein sequences (if the gene is a coding gene), and on whether the inferred phylogenies for the genes correspond with inferred phylogenies of the species where the genes reside.

Sure, lots of complications, but that'll work for now.

SRM said...

photosynthesis, in your first paragraph I think you have the situations reversed. It is generally much easier to conceive as regulatory regions as part of the gene in bacteria as they are generally more compact and contiguous with the transcribed region than they are in eukaryotes (caveats and qualifications notwithstanding).

Larry Moran said...

photosynthesis says,

People annotating genes in eukaryotes tend to consider regulatory regions as part of the gene.

That's not correct. Here's the GenBank entry for one of my favorite genes, HSPA5 on human Chromosome 9.

Here's the RefSeq entry for the same gene.

And here's the UCSC Genome Browser entry for HSPA5

You can see that in all three cases the gene is defined from the transcription start site to the transcription termination site. In other words, the "gene" is the DNA that's transcribed.

anonymous said...

Gene nomenclature can be problematic and practice still remains inconsistent depending on field of expertise.

From an interesting paper:

Developing a community-based genetic nomenclature for anole lizards - Kenro Kusumi , Rob J Kulathinal et al

The salient quote:

“Gene expression
Following gene duplication events, divergence of regulatory control regions can lead to differentiation in tissue specificity and timing of gene expression in paralogous genes. These regulatory regions are considered part of the gene being compared, but it is not straightforward to assign a score to this divergence. Genes that appear to be orthologous by the measures above can still display strikingly different gene expression, raising the question of whether the regulatory gene functionality has diverged in an opposing fashion to that of the protein coding sequence.”

Consensus on a common definition for “gene” remains somewhat elusive.

Continuing on the subject of orthologous relationships, I cite a passage from HUGO that could prove relevant to this continuing series.

“6.5 Pseudogenes

Pseudogenes are sequences that are generally untranscribed and untranslated and which have high homology to identified genes. However, it has recently been shown that in different organisms or tissues functional activation may occur. Therefore, the previous policy of assigning the gene symbol of the structural gene followed by "P" and a number will only be approved on a case by case basis. In future, pseudogenes will usually be assigned the next number in the relevant symbol series, suffixed by a "P" for pseudogene (or "PS" in the specific cases) if requested e.g. OR5B12P "olfactory receptor, family 5, subfamily B, member 12 pseudogene". However, the designation "pseudogene" will remain in the gene name.”

Anonymous said...

Thanks Larry, you're right. I misremembered out of the contrast with gene annotations in prokaryotes.

SRM, while you're right that it would be easier to think of regulatory sites in bacteria as part of the gene, annotations in bacteria start with the first codon, end with the stop codon. (However, some regulatory sites are far, far away, like in eukaryotes, requiring bending of the DNA for several regulatory proteins to touch each other, etc.)

anonymous said...

I hope we are distinguishing between "gene prediction" and "gene annotation"

Unless I am sorely mistaken, the former GENERALLY does not include UTRs where the latter GENERALLY does.