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Wednesday, March 13, 2024

Nils Walter disputes junk DNA: (7) Conservation of transcribed DNA

I'm discussing a recent paper published by Nils Walter (Walter, 2024). He is arguing against junk DNA by claiming that the human genome contains large numbers of non-coding genes.

This is the seventh post in the series. The first one outlines the issues that led to the current paper and the second one describes Walter's view of a paradigm shift/shaft. The third post describes the differing views on how to define key terms such as 'gene' and 'function.' In the fourth post I discuss his claim that differing opinions on junk DNA are mainly due to philosophical disagreements. The fifth and sixth posts address specific arguments in the junk DNA debate.

Sequence conservation

If you don't know what a transcript is doing then how are you going to know whether it's a spurious transcript or one with an unknown function? One of the best ways is to check and see whether the DNA sequence is conserved. There's a powerful correlation between sequence conservation and function: as a general rule, functional sequences are conserved and non-conserved sequences can be deleted without consequence.

There might be an exception to the the conservation criterion in the case of de novo genes. They arise relatively recently so there's no history of conservation. That's why purifying selection is a better criterion. Now that we have the sequences of thousands of human genomes, we can check to see whether a given stretch of DNA is constrained by selection or whether it accumulates mutations at the rate we expect if its sequence were irrelevant junk DNA (neutral rate). The results show that less than 10% of our genome is being preserved by purifying selection. This is consistent with all the other arguments that 90% of our genome is junk and inconsistent with arguments that most of our genome is functional.

This sounds like a problem for the anti-junk crowd. Let's see how it's addressed in Nils Walter's article in BioEssays.

There are several hand-waving objections to using conservation as an indication of function and Walter uses them all plus one unique argument that we'll get to shortly. Let's deal with some of the "facts" that he discusses in his defense of function. He seems to agree that much of the genome is not conserved even though it's transcribed. In spite of this, he says,

"... the estimates of the fraction of the human genome that carries function is still being upward corrected, with the best estimate of confirmed ncRNAs now having surpassed protein-coding genes,[12] although so far only 10%–40% of these ncRNAs have been shown to have a function in, for example, cell morphology and proliferation, under at least one set of defined conditions."

This is typical of the rhetoric in his discussion of sequence conservation. He seems to be saying that there are more than 20,000 "confirmed" non-coding genes but only 10%-40% of them have been shown to have a function! That doesn't make any sense since the whole point of this debate is how to identify function.

Here's another bunch of arguments that Walter advances to demonstrate that a given sequence could be functional but not conserved. I'm going to quote the entire thing to give you a good sense of Walter's opinion.

A second limitation of a sequence-based conservation analysis of function is illustrated by recent insights from the functional probing of riboswitches. RNA structure, and hence dynamics and function, is generally established co-transcriptionally, as evident from, for example, bacterial ncRNAs including riboswitches and ribosomal RNAs, as well as the co-transcriptional alternative splicing of eukaryotic pre-mRNAs, responsible for the important, vast diversification of the human proteome across ∼200 cell types by excision of varying ncRNA introns. In the latter case, it is becoming increasingly clear that splicing regulation involves multiple layers synergistically controlled by the splicing machinery, transcription process, and chromatin structure. In the case of riboswitches, the interactions of the ncRNA with its multiple protein effectors functionally engage essentially all of its nucleotides, sequence-conserved or not, including those responsible for affecting specific distances between other functional elements. Consequently, the expression platform—equally important for the gene regulatory function as the conserved aptamer domain—tends to be far less conserved, because it interacts with the idiosyncratic gene expression machinery of the bacterium. Consequently, taking a riboswitch out of this native environment into a different cell type for synthetic biology purposes has been notoriously challenging. These examples of a holistic functioning of ncRNAs in their species-specific cellular context lay bare the limited power of pure sequence conservation in predicting all functionally relevant nucleotides.

I don't know much about riboswitches so I can't comment on that. As for alternative splicing, I assume he's suggesting that much of the DNA sequence for large introns is required for alternative splicing. That's just not correct. You can have effective alternative splicing with small introns. The only essential parts of introns sequences are the splice sites and a minimum amount of spacer.

Part of what he's getting at is the fact that you can have a functional transcript where the actual nucleotide sequence doesn't matter so it won't look conserved. That's correct. There are such sequences. For example, there seem to be some examples of enhancer RNAs, which are transcripts in the regulatory region of a gene where it's the act of transcription that's important (to maintain an open chromatin conformation, for example) and not the transcript itself. Similarly, not all intron sequences are junk because some spacer sequence in required to maintain a minimum distance between splice sites. All this is covered in Chapter 8 of my book ("Noncoding Genes and Junk RNA").

Are these examples enough to toss out the idea of sequence conservation as a proxy for function and assume that there are tens of thousands of such non-conserved genes in the human genome? I think not. The null hypothesis still holds. If you don't have any evidence of function then the transcript doesn't have a function—you may find a function at some time in the future but right now it doesn't have one. Some of the evidence for function could be sequence conservation but the absence of conservation is not an argument for function. If conservation doesn't work then you have to come up with some other evidence.

It's worth mentioning that, in the broadest sense, purifying selection isn't confined to nucleotide sequence. It can also take into account deletions and insertions. If a given region of the genome is deficient in random insertions and deletions then that's an indication of function in spite of the fact that the nucleotide sequence isn't maintained by purifying selection. The maintenance definition of function isn't restricted to sequence—it also covers bulk DNA and spacer DNA.

(This is a good time to bring up a related point. The absence of conservation (size or sequence) is not evidence of junk. Just because a given stretch of DNA isn't maintained by purifying selection does not prove that it is junk DNA. The evidence for a genome full of junk DNA comes from different sources and that evidence doesn't apply to every little bit of DNA taken individually. On the other hand, the maintenance function argument is about demonstrating whether a particular region has a function or not and it's about the proper null hypothesis when there's no evidence of function. The burden of proof is on those who claim that a transcript is functional.)

This brings us to the main point of Walter's objection to sequence conservation as an indication of function. You can see hints of it in the previous quotation where he talks about "holistic functioning of ncRNAs in their species-specific cellular context," but there's more ...

Some evolutionary biologists and philosophers have suggested that sequence conservation among genomes should be the primary, or perhaps only, criterion to identify functional genetic elements. This line of thinking is based on 50 years of success defining housekeeping and other genes (mostly coding for proteins) based on their sequence conservation. It does not, however, fully acknowledge that evolution does not actually select for sequence conservation. Instead, nature selects for the structure, dynamics and function of a gene, and its transcription and (if protein coding) translation products; as well as for the inertia of the same in pathways in which they are not involved. All that, while residing in the crowded environment of a cell far from equilibrium that is driven primarily by the relative kinetics of all possible interactions. Given the complexity and time dependence of the cellular environment and its environmental exposures, it is currently impossible to fully understand the emergent properties of life based on simple cause-and-effect reasoning.

The way I see it, his most important argument is that life is very complicated and we don't currently understand all of it's emergent properties. This means that he is looking for ways to explain the complexity that he expects to be there. The possibility that there might be several hundred thousand regulatory RNAs seems to fulfil this need so they must exist. According to Nils Walter, the fact that we haven't (yet) proven that they exist is just a temporary lull on the way to rigorous proof.

This seems to be a common theme among those scientists who share this viewpoint. We can see it in John Mattick's writings as well. It's as though the logic of having a genome full of regulatory RNA genes is so powerful that it doesn't require strong supporting evidence and can't be challenged by contradictory evidence. The argument seems somewhat mystical to me. Its proponents are making the a priori assumption that humans just have to be a lot more complicated than what "reductionist" science is indicating and all they have to do is discover what that extra layer of complexity is all about. According to this view, the idea that our genome is full of junk must be wrong because it seems to preclude the possibility that our genome could explain what it's like to be human.

Walter, N.G. (2024) Are non‐protein coding RNAs junk or treasure? An attempt to explain and reconcile opposing viewpoints of whether the human genome is mostly transcribed into non‐functional or functional RNAs. BioEssays:2300201. [doi: 10.1002/bies.202300201]


SPARC said...

If cells are as complex as Walter assumes and all the non-intronic non-coding spurious RNAs were functional and of such regulatory importance then why don't they show up in mouse ENU screens at the same frequencies as mutations in protein coding genes (mutated introns show up more frequently because mis-splicing may interfere with protein expression).
The same is true for gene trap analyses in murine ES cells. For a gene traps to work they just have to be inserted in any kind of transcribed sequence. This approach will only address sequences transcribed in ES cells. However, the only counter argument would be that non-coding RNAs don't play a role in the regulation of ES cells which would be quite a stretch giving the fact that the no-junk proponents often argue that these sequences especially function during development. In addition, if a piece of DNA is only transcribed once in a while in one of thousands cells a gene trap will not work and renders these trapped sequence junk.

Larry Moran said...

@SPARC: I struggle to understand the worldview of the anti-junk crowd. It's seems to me that they have convinced themselves that human (and mouse?) cells are incredibly complex so there must be lots and lots of regulatory sequences, regulatory RNAs, ubiquitous alternative splicing, mysterious functioning transposon fragments, and a complex network of chromatin interactions. All of this is mediated by the dark genome that's full of mysterious undiscovered function.

Normally you would look for evidence to support such a worldview and accept all the nasty little facts that conflict with your hypothesis. That doesn't seem to be happening in this case. Instead, the anti-junk crowd is determined to come up with more and more weird ad hoc excuses to explain away evidence that contradicts their views.