Saturday, June 24, 2017

Debating alternative splicing (part II)

Mammalian genomes are very large. It looks like 90% of it is junk DNA. These genomes are pervasively transcribed, meaning that almost 90% of the bases are complementary to a transcript produced at some time during development. I think most of those transcripts are due to inappropriate transcription initiation. They are mistakes in transcription. The genome is littered with transcription factor binding sites but only a small percentage are directly involved in regulating gene expression. The rest are due to spurious binding—a well-known property of DNA binding proteins. These conclusions are based, I believe, on a proper understanding of evolution and basic biochemistry.

If you add up all the known genes, they cover about 30% of the genome sequence. Most of this (>90%) is intron sequence and introns are mostly junk. The standard mammalian gene is transcribed to produce a precursor RNA that is subsequently processed by splicing out introns to produce a mature RNA. If it's a messenger RNA (mRNA) then it will be translated to produce a protein (technically, a polypeptide). So far, the vast majority of protein-coding genes produce a single protein but there are some classic cases of alternative splicing where a given gene produces several different protein isoforms, each of which has a specific function.

Over the years it has been possible to detect RNA variants that don't correspond to the standard mRNA. Most genes have a large number of such variants. They are deposited in various databases such as the ECgene database (ECgene: an alternative splicing database). Here's an example of splice variants of the human triose phosphate isomerease gene (TPI1).1


Most of these splice variants would produce various isoforms of triose phosphate isomerase if the RNA variant were translated.

There are two explanation of the data ...
Massive alternative splicing: The processing variants represent true biologically functional molecules and the predicted protein products all have a biological function that's unknown at the present time. This gives rise to the meta-claim that almost all human genes are subject to alternative splicing and almost all human genes produce 5-10 different functional protein isoforms. This suggests that there's exquisite regulation of alternative splicing due to splicing factors.

Splicing errors: Most of the variants are due to splicing errors (mistakes). This view is consistent with the fact that the variants are present at very low concentrations and that the error rate of the spliceosome reaction is known to be far less accurate than DNA replication or transcription.
Most of the labs that work on these splice variants are proponents of massive alternative splicing. They operate on the assumption that this phenomenon is a real biological phenomenon and that there are tens of thousands of undiscovered protein isoforms inside most cells.

Most biochemists and molecular biologists accept this explanation. That's why statements like to one below go unchallenged on the Nature science education website (Pray, 2008).
Alternative splicing was the first phenomenon scientists discovered that made them realize that genomic complexity cannot be judged by the number of protein-coding genes. During alternative splicing, which occurs after transcription and before translation, introns are removed and exons are spliced together to make an mRNA molecule. However, the exons are not necessarily all spliced back together in the same way. Thus, a single gene, or transcription unit, can code for multiple proteins or other gene products, depending on how the exons are spliced back together. In fact, scientists have estimated that there may be as many as 500,000 or more different human proteins, all coded by a mere 20,000 protein-coding genes.
The minority view, which I support, is that almost all of those splice variants are due to processing errors and the average protein-coding gene produces a single functional polypeptide.


1. Triose phosphate isomerase is a well-studied enzyme of the gluconeogenesis and glycolysis pathways. It is present in all species.

30 comments :

  1. Just fascinating - may I ask do you think there is a spectrum with splicing errors always producing multiple isoforms but some are useful and hence can be seen by natural selection and hence selected to become more prevalent while others are detrimental and so are selected against and between these two extremes a blur of neutral or nearly isoforms which can exist to a certain extent within a tissue/cell type.

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  2. So, how do you explain the complexity of an organism with much less protein coding genes, like human genome having only 20.000 protein coding genes, in comparison to much less complex eukaryotic organisms with many more protein coding genes, if you through away the alternative splicing?



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    1. Which organisms with many more protein coding genes are you thinking of?

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    2. Why yes, it does. Are there such organisms? What are they? How many genes do they have?

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    3. Uff, the definition of complexity is hard. Although it might seem counterintuitive, complexity does not seem to be related to the number of genes. Although in unicellular organisms like bacteria the number of genes is possibly a good indicator of how many things they can do, in multicellular organisms the number of genes does not tell much. For example, there are plants with much more genes than humans.

      If we compare flies and humans, how would you define which one is more complex? the number of different cell types? If we are to think of intelligence for example, our higher abilities are not related to new genes in the primate lineage. Differences must be explained by different regulation and possibly (I guess) variations in protein-protein interactions and protein kinetics in pre-existing genes.

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    4. Why yes, it does. Are there such organisms?

      You didn't know?

      What are they?

      Do you know of an organism more complex than human?

      How many genes do they have?

      According to which definition of a gene?

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    5. I smell a dog's-ass plot coming

      Just wondering: is your first name Doug?

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    6. There are single-celled organisms with very complex splicing too, for example Bigelowiella natans

      https://www.nature.com/nature/journal/v492/n7427/full/nature11681.html

      And there are others that have double the number exons than humans where splicing hasn't really been studied (for example, humans have on average 9 introns per gene, the two sequenced Symbiodinium genomes both have 18 introns per gene, i.e. double

      Pretty much any complexity feature you can think of that is supposedly unique to humans you can find in much simpler organisms, including single celled ones.

      Large genomes? Ours is only 3GB, but there are vertebrates and plants with 100+GB genomes, and while those are still complex multicellular organisms, single-celled ones have huge genomes too (as a most famous example, dinoflagellates have on average the biggest genomes of all eukaryotes). There is an overall positive correlation between organismal complexity and genome size, but that is probably because of organismal size, not because of complexity itself

      Gene number? Humans have 20,000, lowly Trichomonas has more than 50,000, as do a number of other unicellular species.

      And vice versa -- there are complex multicellular organisms that have few genes. Drosophila is well known to have only 13,000, but there are also fairly complex fungal species with macroscopic fruiting bodies and quite a few cell types that have less than 6,000 genes.

      Etc.

      "Complexity" is an emergent phenomenon, it is fundamentally misguided to look for simple explanations for it.

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    7. Which view do you support on splice variants?

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    8. Do you know of an organism more complex than human?

      Depends on what aspect of biology you are talking about.

      Humans are only "more complex" in their brains, other than that their bodies are in no way more "complex" than those of other mammals, in fact some systems are clearly more complicated in other groups (for example our digestive tract is quite reduced in comparison with herbivorous mammals).

      Then there is development, which is fairly simple in us in comparison to the radical transformations that a holometabolous insect goes through. And those in turn are not as complex as the multiple stages that some parasitic worms go through.

      Complex genomes? Well, ours is fairly messed up because regulatory elements are sparse so you have to have this spaghetti bowl of long-range regulation. But salamanders and lungfish probably have it even worse for the same reasons as theirs are two orders of magnitude bigger and accordingly even sparser.

      Then let's put that aside and compare our genome organization with what you see in spirotrichean ciliates where the germline genome has to be NONLINEARLY spliced into the somatic genome so that it can be expressed. That's an incredibly high-wire act. We do nothing like that.

      Or with the kinetoplastid with its baroque system of RNA editing, guide RNAs, minicircles, etc. Our mitochondrion is just 16.5kb and produces three transcripts.

      Etc.

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    9. What is your point?


      I think it was fairly clear, but let's rephrase it: even alternative splicing is your explanation for mammalian complexity, than why does a lowly single-celled chlorarachniophyte exhibit it too?

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    10. Jass, Are you unable to answer any single question put to you? Or is it just a tactic to deflect attention from problems with your claims?

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    11. Arabidopsis Rhaliana 25.500 protein coding genes

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    12. I asked: "How do you explain complexity if you through away alternative splicing"?

      I didn't say that alternative slicing was my answer to complexity did I?

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    13. Humans are only "more complex" in their brains

      Only? Really...

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    14. Back to the issue: How do you explain mammalian complexity if alternative splicing is not the answer?

      We already know that some organisms use alternative splicing and they are not as complex as mammals...I get it!

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    15. Did you answer all my questions?

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    16. There are single-celled organisms with very complex splicing too, for example Bigelowiella natans

      https://www.nature.com/nature/journal/v492/n7427/full/nature11681.html

      And there are others that have double the number exons than humans where splicing hasn't really been studied (for example, humans have on average 9 introns per gene, the two sequenced Symbiodinium genomes both have 18 introns per gene, i.e. double

      Pretty much any complexity feature you can think of that is supposedly unique to humans you can find in much simpler organisms, including single celled ones.

      Large genomes? Ours is only 3GB, but there are vertebrates and plants with 100+GB genomes, and while those are still complex multicellular organisms, single-celled ones have huge genomes too (as a most famous example, dinoflagellates have on average the biggest genomes of all eukaryotes). There is an overall positive correlation between organismal complexity and genome size, but that is probably because of organismal size, not because of complexity itself

      Gene number? Humans have 20,000, lowly Trichomonas has more than 50,000, as do a number of other unicellular species.

      And vice versa -- there are complex multicellular organisms that have few genes. Drosophila is well known to have only 13,000, but there are also fairly complex fungal species with macroscopic fruiting bodies and quite a few cell types that have less than 6,000 genes.

      Etc.

      "Complexity" is an emergent phenomenon, it is fundamentally misguided to look for simple explanations for it.


      Evolution is mysterious isn't it? But not to believers... to them all is well...I guess it is understandable...

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    17. But, let's turn it around;

      Why would evolution develop so many different ways to evolve complexity? The mechanism is supposedly the same but no more then a handful of evolutionists can agree on one of them...

      Some like natural selection. Some like drift. Others say we need the new mechanism...No wonder complexity is an emergent phenomenon if they are still looking for the mechanism of evolution that is a fact...lol

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    18. The difference between 23,000 and 25,500 does not strike me as very interesting or in need of an explanation. I don't think it compares to the C-value paradox at all. And I think this is all driven by your need to feel special. Get over it.

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  3. Protein coding genes are just raw data

    An astonishing similarity of protein coding genes in humans, pigs, mice, dolphins, kangaroos, spiders etc.

    https://proteomics.cancer.gov/whatisproteomics

    Excerpt: "The human genome contains about 21,000 protein-encoding genes, but the total number of proteins in human cells is estimated to be between 250,000 to one MILLION."

    My comment: Newest studies have confirmed the number of human protein coding genes to be about 19,000. RNA-directed cellular mechanisms are able to build thousands of different proteins by using raw data of one gene, without changing the gene's sequence. Mechanism is called alternative mRNA splicing. Based on DSCAM gene in Drosophila Melanogaster, the fruit fly's cell is able to build even 38,016 different proteins, without changing the sequence. The way of how the cell uses protein coding genes tells us that genes are not drivers. Instead, they are just raw data, libraries for RNA-directed mechanisms.

    http://www.sciencedirect.com/science/article/pii/S0092867400001288


    The alternative splicing is the most significant mechanism inducing the ecological adaptation and organisms' variation. The clever question arises: how come variation of organisms happen if the sequences of protein coding genes are not to be changed? Mutations in protein coding genes could be extremely harmful and they would cause a huge mess in genomic stability and integrity.

    Serious science has the answer:
    Epigenetic Control of Gene Expression. The alternative splicing mechanism is regulated by several epigenetic factors. The three main regulators are:

    1. The DNA methylation.
    http://www.cell.com/trends/genetics/abstract/S0168-9525(15)00040-2

    2. MicroRNA downregulation.
    https://www.lakeforest.edu/live/news/4451-co-regulation-of-mirna-biogenesis-and-alternative

    3. Histone methylation
    https://www.ncbi.nlm.nih.gov/pubmed/27606339

    By the way, did you know that the C. Elegans, a tiny multicellular nematode worm, that is assumed to be a simple type of organism, has 20,450 protein coding genes? More than us! It is not a simple life form! It also uses the alternative splicing machinery for regulating its proteins in 1031 cells it has.

    Protein coding genes are very similar in most animals. For example, human and mice genomes differ only at 2.5%.

    https://www.newscientist.com/article/dn2352-just-2-5-of-dna-turns-mice-into-men/

    Human and pig genomes are also extremely similar.

    http://www.nationalhogfarmer.com/news/human-to-pig

    "We took the human genome, cut it into 173 puzzle pieces and rearranged it to make a pig,” explains animal geneticist Lawrence Schook. “Everything matches up perfectly. The pig is genetically very close to humans."

    Human protein coding genes are also very similar to kangaroos, dolphins, spiders etc. These are very inconvenient facts for believers of the evolutionary theory that is misleading people by maintaining the heresy of human-chimp genomic similarity. They don't tell you that the famous 98% is true only with these protein coding genes. But when we compare the alternative splicing mechanism and epigenomes, the differences are quite remarkable.

    Everything points to Design and Creation. Don't get misled.

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  4. Doesn't the massive alternate splicing explanation require that a very tight correlation between global structure and function in the overwhelming majority of cases? That, in effect, all enzymes are allosteric? It is not clear to me that splicing changes "the" binding site, the coding for which I somehow had the impression was one of the units spliced together for the final product. If the rule is that enzymes are highly allosteric, it is hard for me to see how intermediates could exist at all, much less be part of an evolutionary development.

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  5. How much DNA is devoted to coding for the spliceosome? How conserved are these sequences?

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  6. Georgi marinov.
    A minor point. I agree, as a creationist, that humans have no more complexity in our bodies then anything else. In fact I disagree that any biology is more or less complex then the other. Its very complex and the differences are trivial . Even the differences are just in type and not inferior complexity.
    its a old evolutionist myth, seeing simple evolving into complex, that created the hierarchy of complexity.

    I don't agree our brains are more complex then bugs or baboons.
    Mines not!
    Instead our memory, possibly, is superior to animals and insects.
    However its our immaterial soul, created like god's soul/image, that is the unique origin of our unique intelligence.
    Mine is!

    It would be a creationist happy point to point out our genes are not more or complex. All biology's genes are the same but rearranged. As a creator would do it.

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    1. I don't agree our brains are more complex then bugs or baboons.
      Mines not!


      Oh, Robert. Don't ever change.

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    2. Well, the output of our brains is at least arguably more complex than that of a baboon, a bird or a bollweevil. But does that mean our brains are _genetically_ more complex?

      The whole point about human behaviour is that it's not preprogrammed - we learn from our environment, we store data in our environment in the form of books, and so on. Compare that to a "simpler" animal whose entire behavioural repertoire is stereotyped, fixed, and thus (presumably) programmed somehow by their genes. One could argue that they need _more_ genetic complexity to encode all those different behaviours, whereas humans only need one instruction: "learn from your surroundings".

      Trying to link behavioural complexity to genetic complexity is like trying to rank the importance of scientific papers by counting the number of words in them.

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    3. Peter
      we store everything in our memory. So why not just the memory is the source for all creatures abilities. No DNA needed. No genes needed. indeed why not genes are just pieces of memory. memory bytes like in computers!
      Creatures just might have basic concepts in memory. so a creature can learn to be afraid of what it once was not.
      famous ones are of our dogs getting scared of thunder which wouldn't happen in nature.
      They get phobic but it really shows how they learn.

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