Alternative splicing: function vs noise

This post is about a recent review of alternative splicing published by my colleague Ben Blencowe in the Dept. of Medical Genetics at the University of Toronto (Toronto, Ontario, Canada). (The other author is Jermej Ule of The Francis Crick Institute in London (UK).) They are strong supporters of the idea that alternative splicing is a common feature of most human genes.

I am a strong supporter of the idea that most splice variants are due to splicing errors and only a few percent of human genes undergo true alternative spicing.

This is a disagreement about the definition of "function." Is the mere existence of multiple splice variants evidence that they are biologically relevant (functional) or should we demand evidence of function—such as conservation—before accepting such a claim?

The Function Wars Part VII: Function monism vs function pluralism

This post is mostly about a recent paper published in Studies in History and Philosophy of Biol & Biomed Sci where two philosophers present their view of the function wars. They argue that the best definition of function is a weak etiological account (monism) and pluralistic accounts that include causal role (CR) definitions are mostly invalid. Weak etiological monism is the idea that sequence conservation is the best indication of function but that doesn't necessarily imply that the trait arose by natural selection (adaptation); it could have arisen by neutral processes such as constructive neutral evolution.

The paper makes several dubious claims about ENCODE that I want to discuss but first we need a little background.

Background

The ENCODE publicity campaign created a lot of controversy in 2012 because ENCODE researchers claimed that 80% of the human genome is functional. That claim conflicted with all the evidence that had accumulated up to that point in time. Based on their definition of function, the leading ENCODE researchers announced the death of junk DNA and this position was adopted by leading science writers and leading journals such as Nature and Science.

Let's be very clear about one thing. This was a SCIENTIFIC conflict over how to interpret data and evidence. The ENCODE researchers simply ignored a ton of evidence demonstrating that most of our genome is junk. Instead, they focused on the well-known facts that much of the genome is transcribed and that the genome is full of transcription factor binding sites. Neither of these facts were new and both of them had simple explanations: (1) most of the transcripts are spurious transcripts that have nothing to do with function, and (2) random non-functional transcription factor binding sites are expected from our knowledge of DNA binding proteins. The ENCODE researchers ignored these explanations and attributed function to all transcripts and all transcription factor binding sites. That's why they announced that 80% of the genome is functional.

Happy Darwin Day! 2020

Charles Darwin, the greatest scientist who ever lived, was born on this day in 1809 [Darwin still spurs tributes, debates] [Happy Darwin Day!] [Darwin Day 2017]. Darwin is mostly famous for two things: (1) he described and documented the evidence for evolution and common descent and (2) he provided a plausible scientific explanation of evolution—the theory of natural selection. He put all this in a book, The Origin of Species by Means of Natural Selection published in 1859—a book that spurred a revolution in our understanding of the natural world. (You can still buy a first edition copy of the book but it will cost you several hundred thousand dollars.)

The Function Wars Part VI: The problem with selected effect function

The term "Function Wars" refers to the debate over the meaning of 'function,' especially in the context of junk DNA.¹ That debate intensified in 2012 after the ENCODE publicity campaign that tried to redefine function to mean anything they want as long as it refutes junk DNA. This is the sixth in a series of posts exploring the debate and why it's important, or not. Links to the other five posts can be found at the bottom or this post.

The world is not inhabited exclusively by fools and when a subject arouses intense interest and debate, as this one has, something other than semantics is usually at stake.
Stephen Jay Gould (1982)Much of the discussion seems like quibbling over semantics but I'm reminded of a similar debate over the mode of evolution: is it gradual or punctuated? As Gould pointed out in 1982, there's a serious issue underlying the debate—an issue that shouldn't get lost in bickering over the meaning of 'gradualistic.' The same warning applies here. It's important to determine how much of the human genome is junk and that requires an understanding of what we mean by junk DNA. However, it's easy to get distracted by focusing on the exact meaning of the word 'function' instead of looking at the big picture.

lncRNA nonsense from Los Alamos

A group of scientists at the Los Alamos National Laboratory (Los Alamos, NM, USA) and their collaborators in Vienna (Austria) and Lethbridge (Alberta, Canada) have worked out the structure of Braveheart lncRNA from mice.

Kim, D.N., Thiel, B.C., Mrozowich, T., Hennelly, S.P., Hofacker, I.L., Patel, T.R., Sanbonmatsu, K.Y. (2020) Zinc-finger protein CNBP alters the 3-D structure of lncRNA Braveheart in solution. Nat. Commun. 11:148 [doi: 10.1038/s41467-019-13942-4]

The authors point out in their paper that lncRNAs are difficult to work with and the 3D structures of only a small number have been characterized. There's nothing in the paper about the problems associated with determining the functions of lncRNAs and nothing about the number of lncRNAs except for this brief opening statement: "Long non-coding RNAs (lncRNAs) constitute a significant fraction of the transcriptome ..."

The Three Domain Hypothesis: RIP

The Three Domain Hypothesis died about twenty years ago but most people didn't notice.

The original idea was promoted by Carl Woese and his colleagues in the early 1980s. It was based on the discovery of archaebacteria as a distinct clade that was different from other bacteria (eubacteria). It also became clear that some eukaryotic genes (e.g. ribosomal RNA) were more closely related to archaebacterial genes and the original data indicated that eukaryotes formed another distinct group separate from either the archaebacteria or eubacteria. This gave rise to the Three Domain Hypothesis where each of the groups, bacteria (Eubacteria), archaebacteria (Archaea), and eukaryotes (Eucarya, Eukaryota), formed a separate clade that contained multiple kingdoms. These clades were called Domains.

Are pseudogenes really pseudogenes?

There are many junk DNA skeptics who claim that most of our genome is functional. Some of them have even questioned whether pseudogenes are mostly junk. The latest challenge comes from a recent review in Nature Reviews: Genetics where the authors try to place the burden of proof on those who say that pseudogenes are broken, nonfunctional, genes (Cheetam et al., 2019). The authors of the review try to make the case that we should not label a DNA sequence as a pseudogene until we can prove that it is truly nonfunctional junk.

I'm about to refute this ridiculous stance but first we need a little background.

Remember MOOCs?

We learned back in 2012 that Massive Open Online Courses (MOOCs) were going to transform higher education. People all over the world, especially in underdeveloped nations, would be able to learn from the best university professors while sitting at home in front of their computers. Several companies entered the market with high expectations of earning enormous profits while altruistically educating students who couldn't afford to go to university.

Are introns mostly junk?

There are many reasons for thinking that introns are mostly junk DNA.

1. The size and sequence of introns in related species are not conserved and almost all of the sequences are evolving at the rate expected for neutral substitutions and fixation by drift.
2. Many species have lost introns or reduced their lengths drastically suggesting that the presence of large introns can be detrimental in some cases (probably large populations).
3. After decades of searching, there are very few cases where introns and/or parts of introns have been shown to be essential.
4. Researchers routinely construct intronless versions of eukaryotic genes and they function normally when re-inserted into the genome.
5. Intron sequences are often littered with transposon and viral sequences that have inserted into the intron and this is not consistent with the idea that intron sequences are important.
6. About 98% of the introns in modern yeast (Saccharomyces cerevisiae) have been eliminated during evolution form a common ancestor that probably had about 18,000 introns [Yeast loses its introns]. This suggests that there was no selective pressure to retain those introns over the past 100 million years.
7. About 245/295 of the remaining introns in yeast have been artificially removed by researchers who are constructing an artificial yeast genome suggesting that over 80% of the introns that survived evolutionary loss are also junk [Yeast loses its introns].

The evolution of citrate synthase

Citrate synthase [EC 2.3.3.1] is one of the key enzymes of the citric acid cycle. It catalyzes the joining of acetyl-CoA and oxaloacetate to produce citrate.

acetyl-CoA + H₂O + oxaloacetate → citrate + HS-CoA + H⁺

We usually think of this reaction in terms of energy production since acetyl-CoA is the end product of glycolysis and the citric acid cycle produces substrates that enter the electron transport system leading to production of ATP. However, it's important to keep in mind that the enzyme also catalyzes the reverse reaction.

The "standard" view of junk DNA is completely wrong

I was browsing the table of contents of the latest issue of Cell and I came across this ....

For decades, the miniscule protein-coding portion of the genome was the primary focus of medical research. The sequencing of the human genome showed that only ∼2% of our genes ultimately code for proteins, and many in the scientific community believed that the remaining 98% was simply non-functional “junk” (Mattick and Makunin, 2006; Slack, 2006). However, the ENCODE project revealed that the non-protein coding portion of the genome is copied into thousands of RNA molecules (Djebali et al., 2012; Gerstein et al., 2012) that not only regulate fundamental biological processes such as growth, development, and organ function, but also appear to play a critical role in the whole spectrum of human disease, notably cancer (for recent reviews, see Adams et al., 2017; Deveson et al., 2017; Rupaimoole and Slack, 2017).

Slack, F.J. and Chinnaiyan, A.M. (2019) The Role of Non-coding RNAs in Oncology. Cell 179:1033-1055 [doi: 10.1016/j.cell.2019.10.017]

Cell is a high-impact, refereed journal so we can safely assume that this paper was reviewed by reputable scientists. This means that the view expressed in the paragraph above did not raise any alarm bells when the paper was reviewed. The authors clearly believe that what they are saying is true and so do many other reputable scientists. This seems to be the "standard" view of junk DNA among scientists who do not understand the facts or the debate surrounding junk DNA and pervasive transcription.

Here are some of the obvious errors in the statement.

1. The sequencing of the human genome did NOT show that only ~2% of our genome consisted of coding region. That fact was known almost 50 years ago and the human genome sequence merely confirmed it.
2. No knowledgeable scientist ever thought that the remaining 98% of the genome was junk—not in 1970 and not in any of the past fifty years.
3. The ENCODE project revealed that much of our genome is transcribed at some time or another but it is almost certainly true that the vast majority of these low-abundance, non-conserved, transcripts are junk RNA produced by accidental transcription.
4. The existence of noncoding RNAs such as ribosomal RNA and tRNA was known in the 1960s, long before ENCODE. The existence of snoRNAs, snRNAs, regulatory RNAs, and various catalytic RNAS were known in the 1980s, long before ENCODE. Other RNAs such as miRNAs, piRNAS, and siRNAs were well known in the 1990s, long before ENCODE.

How did this false view of our genome become so widespread? It's partially because of the now highly discredited ENCODE publicity campaign orchestrated by Nature and Science but that doesn't explain everything. The truth is out there in peer-reviewed scientific publications but scientists aren't reading those papers. They don't even realize that their standard view has been seriously challenged. Why?

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The evolution of de novo genes

De novo genes are new genes that arise spontaneously from junk DNA [De novo gene birth]. The frequency of de novo gene creation is important for an understanding of evolution. If it's a frequent event, then species with a large amount of junk DNA might have a selective advantage over species with less junk DNA, especially in a changing environment.

Last week I read a short Nature article on de novo genes [Levy, 2019] and I think the subject deserves more attention. Most new genes in a species appear to arise by gene duplication and subsequent divergence but de novo genes are genes that are unrelated to genes in any other clade so we can assume that they are created from junk DNA that accidentally becomes associated with a promoter causing the DNA to be transcribed. A new gene is formed if the RNA acquires a function. If the transcript contains an open reading frame then it may be translated to produce a polypeptide and if the polypeptide performs a new function then the resulting de novo gene is a new protein-coding gene.

The important question is whether the evolution of de novo genes is a common event or a rare event.

How many protein-coding genes in the human genome? (2)

It's difficult to know how many protein-coding genes there are in the human genome because there are several different ways of counting and the counts depend on what criteria are used to identify a gene. Last year I commented on a review by Abascal et al. (2018) that concluded there were somewhere between 19,000 and 20,000 protein-coding genes. Those authors discussed the problems with annotation and pointed out that the major databases don't agree on the number of gene [How many protein-coding genes in the human genome?].

Gerald Fink promotes a new definition of a gene

This is the 2019 Killian lecture at MIT, delivered in April 2019 by Gerald Fink. Fink is an eminent scientist who has done excellent work on the molecular biology of yeast. He was director of the prestigious Whitehead Institute at MIT from 1990-2001. With those credentials you would expect to watch a well-informed presentation of the latest discoveries in molecular genetics. Wouldn't you?

Contingency, selection, and the long-term evolution experiment

I'm a big fan of Richard Lenski's long-term evolution experiment (LTEE) and of Zachary Blount's work in particular. [Strolling around slopes and valleys in the adaptive landscape] [On the unpredictability of evolution and potentiation in Lenski's long-term evolution experiment] [Lenski's long-term evolution experiment: the evolution of bacteria that can use citrate as a carbon source]

The results of the LTEE raise some interesting questions about evolution. The Lenski experiment began with 12 (almost) identical cultures and these have now "evolved" for 31 years and more than 65,000 generations. All of the cultures have diverged to some extent and one of them (and only one) has developed the ability to use citrate as a carbon source. Many of the cultures exhibit identical, or very similar, mutations that have reached significant frequencies, or even fixation, in the cultures.

Several other laboratory evolution experiments have been completed or are underway in various labs around the world. The overall results are relevant to a discussion about the role of contingency and accident in the history of life [see Evolution by Accident]. Is it true that if you replay the tape of life the results will be quite different? [Replaying life's tape].