The spliceosome: a molecular machine that defies any non-design explanation.

_{Giuseppe Puccio

December 21, 2017

Intelligent Design}

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OK, let’s start with a very simple fact: eukaryotic genes have introns.

IOWs, they are not continuous. They are made of exons and introns: exon – intron – exon – intron – exon and so on. Exons code for the protein. Introns don’t.

So, when the content of the gene is copied to the mRNA, introns must be cut away, and only exons are retained, in order to be translated, so that the mature mRNA can be transferred to the cytoplasm and translated by the ribosome.

This process of removing introns is called splicing.

Now, a few clarifications:

a) Introns exist in prokaryotes too, but they are rather rare. For our purposes, we will only discuss introns in eukaryotes.

b) Introns exist in many different types of genes. For our purposes, we will discuss only those in protein coding genes.

c) The origin and possible function of introns is, still, a mystery.

d) Introns are usually longer than exons. In humans, for example, they amount to approximately 35% of the whole geneome, vs about 1.5% of coding exons.

e) However, the amount and length of introns can vary a lot in different organims. An extreme example is yeast (s. cerevisiae), whose genome contains a very small amount of introns (about 250 out of about 6250 genes).

When the gene if transcribed, both exons and introns are transcribed. A 5′ UTR (Untranslated region) and 3′ UTR, is also part of the pre-mRNA.

Fig. 1

Pre-mRNA is the first form of RNA created through transcription in protein synthesis. The pre-mRNA lacks structures that the messenger RNA (mRNA) requires. First all introns have to be removed from the transcribed RNA through a process known as splicing. Before the RNA is ready for export, a Poly(A)tail is added to the 3’ end of the RNA and a 5’ cap is added to the 5’ end. – By Nastypatty (Own work) [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)], via Wikimedia Commons

So the question is: how are introns removed from pre-mRNA? IOWs, how is splicing achieved?

And there is more. As everybody probably knows, splicing is not always done in the same way. Different isoforms of the same protein can be obtained by alternative splicing, and they can have functional differences. I will not go into details about that, but here is the Wikipedia page about alternative splicing:

https://en.wikipedia.org/wiki/Alternative_splicing

So, how is splicing done, and how is alternative splicing regulated?

We know much about the first question, very little about the second.

There are three ways to perform splicing:

Spliceosomal splicing
Self-splicing
tRNA splicing

The last two modalities are rare, and can be found both in prokaryotes and eukaryotes. I will not discuss them here.

So, the subject of this OP is spliceosomal splicing, which is restricted to eukaryotes.

Moreover, I will discuss only the major spliceosome, which is responsible for the vast majority of splicing in eukaryotes. It must be said, however, that also a minor spliceosome exists, and that it acts in a minority of cases.

So, the spliceosome.

The first important point is:

It is an amazing molecular machine. Even more, it is an amazing molecular cycle, involving many different stages each of which is an amazing molecular machine.

Let’s see. Here is a figure which summarizes the main stages of the spliceosome cycle:

Fig. 2

Spliceosomal splicing cycle – By JBrain [CC BY-SA 2.0 de (https://creativecommons.org/licenses/by-sa/2.0/de/deed.en)], via Wikimedia Commons

To make it simple, the spliceosome units are built upon 5 specific RNAs, called small nuclear RNAs (snRNA). These are, in humans:

U1 (164 bases), U2 (187 bases), U4 (145 bases), U5 (116 bases), U6 (107 bases)

They are transcribed from multiple gene copies. While their sequences are not particularly conserved (U6 being the most conserved of all), their secondary structure seems to be very conserved.

snRNAs are very important in the spliceosome, because they seem to be responsible for the catalytic activities.

Each of the 5 snRNAs forms a complex with proteins, and the complex takes the name of snRNP. The whole spliceosome includes at least 145 different proteins, maybe more, some of which are still not well known.

I will mention here some of the most important:

U1 snRNP:

U1 snRNP 70 kDa (P08621, 437 AAs)

U1 snRNP A (P09012, 282 AAs)

U1 snRNP C (P09234, 159 AAs)

Sm proteins: 7 small proteins (76 – 240 AAs) which form the “Sm core” ring in spliceosome subunits U1, U2, U4, U5, a ring which hosts the specific snRNA molecule.

U2 snRNP:

U2 snRNP A’ (P09661, 255 AAs)

U2 snRNP B” (P08579, 225 AAs)

SF3a120 (Q15459, 793 AAs)

SF3a66 (Q15428, 464 AAs)

SF3a60 (Q12874, 501 AAs)

SF3b155 (O75533, 1304 AAs)

SF3b145 (Q13435, 895 AAs)

SF3b130 (Q15393, 1217 AAs)

SF3b49 (Q15427, 424 AAs)

SF3b14a/p14 (Q9Y3B4, 125 AAs)

SF3b10 (Q9BWJ5, 86 AAs)

Sm proteins

U4/U6 snRNP:

Prp3 (O43395, 683 AAs)

Prp31 (Q8WWY3, 499 AAs)

Prp4 (O43172, 522 AAs)

Cyph ( O43447, 177 AAs)

15.5 K (P55769, 128 AAs)

Sm proteins (for the U4 snRNA)

Lsm proteins: a number of proteins similar to Sm proteins (usually Lsm 2-8), which form a specific ring for the U6 snRNA.

U5 snRNP:

Prp8 (Q6P2Q9, 2335 AAs)

BRR2 (O75643, 2136 AAs)

Snu114 (Q15029, 972 AAs)

Prp6 (O94906, 941 AAs)

Prp28 (Q9BUQ8, 820 AAs)

52 K (O95400, 341 AAs)

40 K (Q96DI7, 357 AAs)

Sm proteins

Additional proteins in the U4/U6/U5 complex:

hSnu66 (O43290, 800 AAs)

hSad1 (Q53GS9, 565 AAs)

27 K (Q8WVK2, 155 AAs)

OK, these are only the main components, and the best understood. We are still far from the sum total of 145/150 proteins which are involved in the spliceosome cycle.

But how does it work?

Always in brief. Here is a typical exon-intron structure:

Fig. 3

Parts of an intron – By miguelferig (Own work) [Public domain], via Wikimedia Commons

GU, A and AG are nucleotides almost universally conserved in all introns, approximately at the positions shown in the figure, and which have a fundamental role in splicing. However, the real stuff is much more complex than this (see the splicing code section). GU (n. 4 in the figure) is near to the 5′ end of the intron, AG (n. 1) near to the 3′ end. The A (n. 3 in the figure) is called “the branch point”. The py-py-py (n. 2 in the figure) is the “polypyrimidine tract”.

a) The U1 subunit binds to the GU sequence at the 5′ splice site in the intron

b) The U2 subunit binds to the “branch point”.

c) The U4/U6/U5 binds to the complex.

d) Numeorus further modifications take place, causing the formation of a “lariat” (including the intron), which is then cleaved, while the two exons are ligated.

I will spare you the many complexities in all the various steps, which are well summarized (in a very simplified way) in this Wikipedia page:

RNA splicing

See in particular the “Formation and activity” section.

Or, if you like more detail, here:

Spliceosome Structure and Function

And here is a very good video on the whole splicing process in yeast:

Now, if somebody still has doubts about the complexity of this molecular machine/process, let’s consider some important aspects.

1. The spliceosome is a molecular machine which appears in eukaryotes.

I quote from this paper (in the abstract):

Origin and evolution of spliceosomal introns

There is no indication that any prokaryote has ever possessed a spliceosome or introns in protein-coding genes, other than relatively rare mobile self-splicing introns.

The following Table shows how some of the main proteins involved in the spliceosome activity show practically no trace of homology in prokaryotes. I have included also included in the table two examples which show low homologies due to some domain which is already expressed in prokaryotes: Prp4, whose 209 bits of homology are due to a specific domain, WD40, and Prp28, which exhibits 313 bits linked to the DEXDc domain. The point is: many of the spliceosome proteins are complete novelties in eukaryotes, but not all of them.

Protein	Bacteria	Archea
U1 snRNP 70 kDa (P08621, 437 AAs)	67	–
U2 SF3b130 (Q15393, 1217 AAs)	43.5	–
U4/U6 hPrp3 (O43395, 683 AAs)	–	–
U4/U6/U5 hSnu66 (O43290, 800 AAs)	–	–
U5 Prp8 (Q6P2Q9, 2335 AAs)	–	–
U5 Prp6 (O94906, 941 AAs)	100	–
U4/U6 hPrp4 (O43172, 522 AAs)	209	150
U/5 Prp28 (Q9BUQ8, 820 AAs)	313	286

I will analyze in more detail one of the most important proteins in the spliceosome, Prp8, in the last part of this OP

(Just a technical note: if you blast Prp8, you will find 3 hits which are obviously an error due to unverified sequences, probably cases of contamination).

2. The spliceosome is a molecular machine which is universally present in eukaryotes.

All eukaryotes have introns, even if in very different degrees, and the spliceosome, even if in some organisms parts of the spliceosome complex can be lost. For a more detailed discussion, look at the Rogozin paper quoted above (Origin and evolution of spliceosomal introns), in particular the section:

Intron density, size and distribution in protein coding genes across the eukaryote domain

I quote this important conclusion:

As pointed out above, despite the existence of numerous, diverse intron-poor genomes, eukaryotes do not lose the “last” intron or the spliceosome although degradation of the spliceosome including loss of many components does occur, e.g. in yeast. The only firmly established exception is the tiny genome of a nucleomorph (an extremely degraded intracellular symbiont of algae) that has lost both all the introns and the spliceosome [7]; preliminary genomic data indicate that all introns might have been lost also in a microsporidium, a highly degraded intracellular parasite distantly related to Fungi [54].

So, whe can conclude that both introns and the spliceosome are a universal feature of eukaryotes, the few exceptions being simply cases of loss of information.

3. The spliceosome is a molecular machine whose information is extremely conserved throughout eukaryotes, up to humans.

I have already mentioned that the 5 RNAs which form the core of the spliceosome are not extremely conserved at sequence level, even if they are extremely conserved at structure level.

However, many of the proteins that compose the spliceosome show an amazing sequence conservation throughout eukaryotes. Now, even if we cannot be certain of when eukaryoyes really emerged, and of their early evolutionary history (both issues being at present highly controversial), we can reasonably assume that protein sequences which are highly conserved in all eukaryotes have been conserved for something like 2 billion years (more or less). As anybody who has followed my previous OPs about information conservation in vertebrates certainly knows, that is an evolutionary time frame which certainly allows us to equal conservation to extremely high functional constraint.

But how conserved are spliceosome proteins? We can analyze a few of them with my usual methodology: looking at human conserved information. The results shows that many proteins involved in the spliceosome are amazingly conserved in all eukaryotes. While there are a few cases which have a rather different evolutionary history, this is by far the most common behaviour for spliceosomal proteins.

Here is a sample of some important sequences that show high homology with the human form in all major groups of single celled eukaryotes. The 5 groups of single celled organisms chosen here, indeed, cover rather well the whole range of single celled eukaryotes.

Fig. 4

These four important proteins, as shown, have an amazing amount of information shared with the human form, ranging from more than 1000 to more than 4000 bits. In bits per AA, the range goes from 0.88 to 1.80 bits per aminoacid (baa). As can be seen, the highest homology is found in fungi, as expected, because fungi are the most likely ancestors of metazoa. The lowest homologies are observed in Naegleria (Excavata) or in Alveolata.

Of course, these proteins remain highly similar to the human form in the following evolutionary history in Metazoa.

So, we can safely state that most spliceosomal proteins, while emerging almost entirely in eukaryotes and showing only trivial homologies with prokaryotes, were probably already universally present in the Last Universal Eukaryotic Ancestor (LECA), and in a form already very similar at sequence level to what we observe in metazoa and in humans.

4. The spliceosome is a wonderful example of irreducibly complexity.

OK, we have already said that splicing can be achieved in at least three different ways. For example, bacterial introns, although rare, are of the self-splicing type. So, we know that the generic function of splicing introns can be implemented in different ways.

But eukaryotic introns are of the spliceosomal type, and they are spliced only by the spliceosome.

We have also said that a minor spliceosome also exists. It shares some featues with the major spliceosome, but it is a different structure and acts on different, and much rarer, introns.

So, for the vast majority of eukaryotic introns, the major spliceosome, and only the major spliceosome, can effectively accomplish the splicing.

Now, I don’t mean here that the major spliceosome must always be absolutely complete, with all its 150 proteins, to be able to work. That’s not what I mean when I say that it is irreducibly complex.

Maybe in some organisms the spliceosome can be partially defective, and still work. It is difficult to say, because we still don’t understand the role of all the components of the spliceosome.

But however, as far as we can understand, most of the principal features must be present, because, as we have seen, the splicing is the result of a complex cycle, involving the RNAs and the subunits, and all stages are essential to the final result.

So, the spliceosome is certainly highly irreducibly complex, even if we may not be able to clearly identify the essential nucleus of molecules which is absolutely necessary to the minimal function.

Moreover, the spliceosome would be useless if spliceosomal introns did not exist, with their properties and code (see later), and spliceosomal introns could not exist if the spliceosome were not there to splice them, because otherwise transcription would be completely ineffective. So, in that sense, spliceosomal introns and the spliceosome are a good example of chicken egg paradox, or we could also say that they form an irreducibly complex system at a higher level. And remember, the whole system seems to have been already present, very much similar to its current form in humans, in LECA, as we have seen.

But, of course, our darwinist friends will simply say that they have co-evolved! 🙂

Moreover, we can and should ask ourselves: why is the spliceosome so complex? The answer is not easy, because we still understand very little, but it is certainly related to the complexity of the splicing code, and to the fundamental issue of alternative splicing.

The Splicing code.

To splice introns by localizing 5 (or a few more) conserved nucleotides and then cutting at the ends of a lariat and rejoining the two exons is certainly a complex task, but apparently not so complex that one of the biggest and most impressing known molecular machines is needed for that. But the simple truth is that recognizing and appropriately splicing all introns is a much more complex task than that.

That is due to the simple fact that conserved nucleotides at the ends are not a sufficient signal to identify the segment that has to be spliced, and that a lot of other components (not always well understood) are necessary to that, and that the splicing is not made always in the same way, and that alternative splicing is a very powerful tool for transcription regulation.

The subject is very complex, and I will not deal with it in depth. However, Those interested could look at this recent review:

The splicing code.

Unfortunately, the paper is paywalled, but the abstract is very informative.

The complexity of the splicing code can give us some insight about the true reasons for the complexity of the spliceosome, and definitely supposrts the idea that the whole system, introns, splicing code and spliceosome, is irreducibly complex.

The Prp8 protein.

I will add a few words about this proteins, which is probably the most amazing component of the spliceosome, and well represents its essential features.

This protein has many amazing charactertistics:

a) It is, as far as I can say, the longest protein in the whole spliceosomal system, and a very long protein indeed: 2335 AAs.

b) It is completely absent in prokaryotes.

c) It is extremely conserved in eukaryotes, probably the most conserved protein in the whole spliceosomal complex (see Fig. 4). In Naegleria, we have 3345 bits of homology with the human protein, corresponding to 1.4 baa, while in fungi we have the amazing result of 4211 bits of human-conserved information, corresponding to 1.8 baa. IOWs, in fungi we already find almost 90% of the functional information present in human Prp8 (remember, the highest possible bitscore is about 1.2 baas), corresponding to 1995 identities (86%) and 2155 positives (92%): a result which is incredibly rare, considering that such information has been conserved for about 2 billion years.

Just to emphasize the importance of this fact, here is the blast between the human protein and the best hit in fungi (Basidiobolus meristosporus):

d) It is a protein which is extremely important for the function of the spliceosome. Here is a very good paper about this protein and its functional relevance:

Prp8 protein: At the heart of the spliceosome

I quote from the conclusions:

Prp8p is central to the expression of all nuclear intron-containing mRNAs. In higher eukaryotes, it is responsible for processing thousands of transcripts in alternative splicing pathways, and in both U2 and U12 spliceosomes. It is important for the pathology of human disease, as all eukaryotic pathogens and parasites require Prp8p to functionally express their genes, in some cases via the trans-spliceosome. Retinitis Pigmentosa, a human genetic disorder that causes progressive blindness, positions Prp8p as a target for therapeutic medicine.

Moreover, another way to assess the functional constraints of a protein is to check its tolerance to polymorphisms in humans. That can be done consulting a very recent and useful database, the ExAC browser, which reports data from about 60000 human genomes.

ExAC gives two important metrics to assess how much a protein is tolerant to polymorphisms and variants. I quote here from the site FAQ:

What are the constraint metrics?

For synonymous and missense, we created a signed Z score for the deviation of observed counts from the expected number. Positive Z scores indicate increased constraint (intolerance to variation) and therefore that the gene had fewer variants than expected. Negative Z scores are given to genes that had a more variants than expected.

For LoF, we assume that there are three classes of genes with respect to tolerance to LoF variation: null (where LoF variation is completely tolerated), recessive (where heterozygous LoFs are tolerated), and haploinsufficient (where heterozygous LoFs are not tolerated). We used the observed and expected variants counts to determine the probability that a given gene is extremely intolerant of loss-of-function variation (falls into the third category). The closer pLI is to one, the more LoF intolerant the gene appears to be. We consider pLI >= 0.9 as an extremely LoF intolerant set of genes.

Now, for Prp8 we get the following results:

Constraint from ExAC	Expected no. variants	Observed no. variants	Constraint Metric
Synonymous	366.6	460	z = -3.02
Missense	920.2	266	z = 10.55
LoF	92.6	8	pLI = 1.00

That means the following:

a) The number of observed missense variants is so low vs expected (266 vs 920.2) that the z value is 10.55 (IOWs, 10.55 standard deviations, more than 10 sigma). Believe me, this is a really exceptional result, most proteins are much more tolerant to missense variants.

b) The probability of loss of function is 1. That means that even heterozygous LoF is absolutely not tolerated.

This is an extremely functional molecule, if ever there was one!

A brief conclusion.

To sum up the meaning of this rather long discussion, I would simply say:

The introns – spliceosome system is a molecular machine of amazing complexity. I have touched only its main aspects in this OP, but believe me, there are layers of complexity there that would require a whole treatise and that I have not even started to mention here.
Whoever can really believe that all this can be explained by some RV + NS model is, IMO, really admirable for his faith in a wrong paradigm.

Comments

gpuccio, That's right, this year is practically over. Let's wait and see...Dionisio_{December 30, 2017
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Dionisio: "Does this relate (somehow) to your concept of biological “procedures”?" Yes, definitely! "Do we have to wait until next year to read a new comment from professor Arthur Hunt?" Don't be impatient. Next year is not so far away! :)gpuccio_{December 30, 2017
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Do we have to wait until next year to read a new comment from professor Arthur Hunt?Dionisio_{December 30, 2017
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gpuccio @253:
So, epigenetics is supposed to supra-regulate transcription, alternative splicing, and many other regulatory networks. We could probably add alternative polyadenilation of mRNA precursors, which seems to be Arthur Hunt’s very interesting main field of research.
The plot thickens...
As I said, we never know enough! But, in the end, the final question always remains: Quis custodiet ipsos custodes? (Giovenale) Who Watches the Watchmen? (Alan Moore) IOWs, what supraregulates epigenetics?
Does this relate (somehow) to your concept of biological "procedures"?Dionisio_{December 30, 2017
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ET at #248 (and all interested): "The one thing we have overlooked is the fact that both splicing and editing require knowledge– meaning they just don’t happen. There has to be knowledge of what to splice and edit- on how to splice and edit- etc" Great point. For example, while I was discussing the psaA trans-splicing here, I have wondered many times about the folowing: How can any system, evolved or not, keep trace of where the different parts are written? Or at least of what the different parts are? Because that seems a basic requirement to recognize them and to reconstruct the fragmented gene. Now, in this case we have 4 parts: the three exons with their flanking fragments of the intron, and the tscA gene. They are probably even degenerate: the tscA RNA certainly needs processing before joining the rest. That reminds me a little of hard disk fragmentation, in a sense! You can write a file fragmenting it in many parts. But you certainly need some record of where the parts are, and of what they are. Now, the point is that the fragmentation and degeneration of the components of the psaA gene are usually attributed to "rearrangements of organelle genomes". IOWs, they should be just unlucky events randomly caused by other DNA rearrangements. One argument in favour of the random origin of the fragmentation could be the simple fact that in similar organisms the same gene is not fragmented. But if the fragmentation is due to some deleterious random event, why should there be any trace of it? Of the nature and location of the fragments? Maybe the location is not important, after all. Trans-splicing occurs on pre-mRNAs, and the location of the gene could not be important. But certainly, the RNA fragments must be recognized, and there must be some trace of how to process and reunite them. As the gene is a fundamental one, it seems that the rescuing information should have existed before the fragmentation event, or just have been created immediately after the event itself. Indeed, the fragmentation events cannot realistically be imagined as gradual. It probably rather happened suddenly, as the result of some major DNA rearrangement. And the organism cannot reasonably avoid strong negative selection, if the gene does not work. I think that's the reason why neo-darwinists recur to the bizarre idea of Constructive Neutral Evolution to make some sense of it: the information was certainly there before the event, kindly provided by random neutral events! :) But, if you read carefully the description of what CNE should be, for example here: http://onlinelibrary.wiley.com/doi/10.1002/iub.489/abstract
If an autonomously functioning cellular component acquires mutations that make it dependent for function on another, pre-existing component or process, and if there are multiple ways in which such dependence may arise, then dependence inevitably will arise and reversal to independence is unlikely.
As you can see, the idea seems to be that "mutations", maybe not too severe, can be balanced by some existing structure, and that the complexity is built "gradually". But there is nothing gradual in the fragmentation of a gene in 4 different parts! How can any pre-existing structure be "ready" to assist some gene if it is randomly fragmented? The idea itself is really beyond my wildest imagination!gpuccio_{December 30, 2017
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Dionisio (and all interested): Very interesting paper at #251. Of course, all regulatory processes with the "alternative" qualification must be supra-regulated by epigenetic changes. Indeed, if something can be done in one way or in some other, alternative way, there must be a reason fro that choice. Of course it could also be a merely random choice, but in that case we could scarcely expect working systems at all. So, epigenetics is supposed to supra-regulate transcription, alternative splicing, and many other regulatory networks. We could probably add alternative polyadenilation of mRNA precursors, which seems to be Arthur Hunt's very interesting main field of research. This is a recent review: Alternative polyadenylation of mRNA precursors https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5483950/
Abstract Alternative polyadenylation (APA) is an RNA-processing mechanism that generates distinct 3? termini on mRNAs and other RNA polymerase II transcripts. It is widespread across all eukaryotic species and is recognized as a major mechanism of gene regulation. APA exhibits tissue specificity and is important for cell proliferation and differentiation. In this Review, we discuss the roles of APA in diverse cellular processes, including mRNA metabolism, protein diversification and protein localization, and more generally in gene regulation. We also discuss the molecular mechanisms underlying APA, such as variation in the concentration of core processing factors and RNA-binding proteins, as well as transcription-based regulation.
And this is probably the most recent paper published by our kind contributor about that: Noncanonical Alternative Polyadenylation Contributes to Gene Regulation in Response to Hypoxia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5502444/
ABSTRACT Stresses from various environmental challenges continually confront plants, and their responses are important for growth and survival. One molecular response to such challenges involves the alternative polyadenylation of mRNA. In plants, it is unclear how stress affects the production and fate of alternative mRNA isoforms. Using a genome-scale approach, we show that in Arabidopsis thaliana, hypoxia leads to increases in the number of mRNA isoforms with polyadenylated 3? ends that map to 5?-untranslated regions (UTRs), introns, and protein-coding regions. RNAs with 3? ends within protein-coding regions and introns were less stable than mRNAs that end at 3?-UTR poly(A) sites. Additionally, these RNA isoforms were underrepresented in polysomes isolated from control and hypoxic plants. By contrast, mRNA isoforms with 3? ends that lie within annotated 5?-UTRs were overrepresented in polysomes and were as stable as canonical mRNA isoforms. These results indicate that the generation of noncanonical mRNA isoforms is an important feature of the abiotic stress response. The finding that several noncanonical mRNA isoforms are relatively unstable suggests that the production of non-stop and intronic mRNA isoforms may represent a form of negative regulation in plants, providing a conceptual link with mechanisms that generate these isoforms (such as alternative polyadenylation) and RNA surveillance.
As I said, we never know enough! :) But, in the end, the final question always remains: Quis custodiet ipsos custodes? (Giovenale) Who Watches the Watchmen? (Alan Moore) IOWs, what supraregulates epigenetics? :)gpuccio_{December 30, 2017
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ET @250: I understand their condition, because I've been there, done that. Unfortunately, as we grow older, we lose our sense of wonder. We stop paying attention to details. Eventually we don't care about the contextual meaning of words. Our condition degenerates to the point that we don't understand the meaning of our experiences because we don't care about it. Then, we don't care about factual evidences and we don't pursue truth. It's a miserable condition that could be described as "anyway, whatever". No amount of academic education or intellectual capacity can free one from such a pathetic condition. What is amazing is that I'm not there doing that anymore. Now I enjoy watching my little grandchildren being fascinated with little things. I pray that they don't lose that beautiful sense of wonder. I would like to teach them to test everything and hold what is good. But that doesn't mean to try everything. How can they test without trying? They'll need wisdom. Where can they get it from? That's what I want to show them.Dionisio_{December 30, 2017
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Connecting the dots: chromatin and alternative splicing in EMT Jessica A. Warns, James R. Davie and Archana Dhasarathy Biochem Cell Biol. 2016 Feb; 94(1): 12–25. doi: 10.1139/bcb-2015-0053 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4704998/pdf/nihms708121.pdfDionisio_{December 30, 2017
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Dionsio- It's amazing what evolutionists have to ignore in order to remain loyal to the blind watchmaker. If ever there was a concept based on ignorance it is evolution by means of blind and mindless processesET_{December 30, 2017
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ET @248: Exactly. What, how, when, why?Dionisio_{December 30, 2017
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The one thing we have overlooked is the fact that both splicing and editing require knowledge- meaning they just don't happen. There has to be knowledge of what to splice and edit- on how to splice and edit- etcET_{December 30, 2017
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#242 Gpuccio Very interesting :) Thanks for giving a review of whats behind the wall. #239 Dionisio, sad days when scientist must worry their careers are in jeopardy for daring to question Darwinism and/or conjecture of finding Design. Take religion out of it. Simply recognizing Design is grounds for firing, run out of office, locked out, entire careers over. The Orwellian Thought Minders seek to punish, not just oppress free thought. So much for "free thinkers." What's so radical about seeing design? In this instance, the 1st Intron? It's astonishing to dismiss it out of hand without serious consideration. It immediately reminded me of hardware/software storage requirements for media, search and information exchange. Can there be some criticisms elsewhere? Sure there can. But most storage and retrieval methods require formation of "marks" and/or "introns" for dynamic access, including headers, pointers and significant indexing. All interspersed through out the "randomly" distributed Metadata. There are different requirements based upon load distribution and purpose of systems. Thus differences across prokaryotes and eukaryotes for splicing and/or storage/retrieval access methods. #245 Diniosio, thanks, will review! Was hoping to read the latest before running into paywall, but these will get me up to speed.DATCG_{December 29, 2017
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Discussion between AH and GP Index of posted comments: AH @25 …….……. GP @28 AH @50 AH @51 …….……. GP @54 AH @56 * …….……. GP @60 …….……. GP @69 …….……. GP @75 …….……. GP @86 …….……. GP @98 …….……. GP @106 …….……. GP @118 …….……. GP @127 …….……. GP @129 AH @130 …….……. GP @136 …….……. GP @138 …….……. GP @146 …….……. GP @162 AH @164 …….……. GP @167 …….……. GP @176 …….……. GP @182 …….……. GP @198 …….……. GP @200 …….……. GP @201 …….……. GP @210 …….……. GP @211 …….……. GP @212 …….……. GP @231 …….……. GP @242 AH is the distinguished professor Arthur Hunt GP is the author of the excellent OP that started this discussion thread (*) first publicly admitted mistake @56 (to be continued…)Dionisio_{December 29, 2017
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The same authors of the 2017 paper referenced by gpuccio @242 coauthored the 2014 papers referenced @243 & @244. The newer paper may contain new information obtained in the 3 years after the older papers were published.Dionisio_{December 29, 2017
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The Supraspliceosome — A Multi-Task Machine for Regulated Pre-mRNA Processing in the Cell Nucleus Author links open overlay panel Kinneret Shefer a, Joseph Sperling a, Ruth Sperling b https://doi.org/10.1016/j.csbj.2014.09.008 Computational and Structural Biotechnology Journal Volume 11, Issue 19, September 2014, Pages 113-122 https://www.sciencedirect.com/science/article/pii/S2001037014000348 https://www.sciencedirect.com/science/article/pii/S2001037014000348/pdfft?md5=21d9c3c06c61fa29987e17e840819804&pid=1-s2.0-S2001037014000348-main.pdf https://pdfs.semanticscholar.org/f461/418c3e6be7f3b809adb84ab5cede7d1c9007.pdfDionisio_{December 29, 2017
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When isolated from mammalian cell nuclei, all nuclear pre-mRNAs are packaged in multi-subunit large ribonucleoprotein complexes—supraspliceosomes—composed of four native spliceosomes interconnected by the pre-mRNA. Supraspliceosomes contain all five spliceosomal U snRNPs, together with other splicing factors, and are functional in splicing. Supraspliceosomes studied thus far represent the steady-state population of nuclear pre-mRNAs that were isolated at different stages of the splicing reaction. To analyze specific splicing complexes, here, we affinity purified Pseudomonas aeruginosa phage 7 (PP7)-tagged splicing complexes assembled in vivo on Adenovirus Major Late (AdML) transcripts at specific functional stages, and characterized them using molecular techniques including mass spectrometry. First, we show that these affinity purified splicing complexes assembled on PP7-tagged AdML mRNA or on PP7-tagged AdML pre-mRNA are assembled in supraspliceosomes. Second, similar to the general population of supraspliceosomes, these defined supraspliceosomes populations are assembled with all five U snRNPs at all splicing stages. This study shows that dynamic changes in base-pairing interactions of U snRNA:U snRNA and U snRNA:pre-mRNA that occur in vivo during the splicing reaction do not require changes in U snRNP composition of the supraspliceosome. Furthermore, there is no need to reassemble a native spliceosome for the splicing of each intron, and rearrangements of the interactions will suffice.
Supraspliceosomes at Defined Functional States Portray the Pre-Assembled Nature of the Pre-mRNA Processing Machine in the Cell Nucleus Hani Kotzer-Nevo 1?, Flavia de Lima Alves 2?, Juri Rappsilber 2,3?, Joseph Sperling 4? and Ruth Sperling 1,* ? Int. J. Mol. Sci. 2014, 15(7), 11637-11664; doi:10.3390/ijms150711637 http://www.mdpi.com/1422-0067/15/7/11637/pdf http://www.mdpi.com/1422-0067/15/7/11637/htm http://www.mdpi.com/1422-0067/15/7/11637

Dionisio_{December 29, 2017
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Arthur Hunt (and all interested): Just to demonstrate that we never know enough. A recent paper, signaled by Dionisio at #192, seems to revolutionize, in some measure, what we know of the spliceosome: Structural studies of the endogenous spliceosome – The supraspliceosome http://www.sciencedirect.com/science/article/pii/S1046202316305060?via%3Dihub Paywalled. Here is the abstract:
Pre-mRNA splicing is executed in mammalian cell nuclei within a huge (21 MDa) and highly dynamic molecular machine – the supraspliceosome – that individually package pre-mRNA transcripts of different sizes and number of introns into complexes of a unique structure, indicating their universal nature. Detailed structural analysis of this huge and complex structure requires a stepwise approach using hybrid methods. Structural studies of the supraspliceosome by room temperature electron tomography, cryo-electron tomography, and scanning transmission electron microscope mass measurements revealed that it is composed of four native spliceosomes, each resembling an in vitro assembled spliceosome, which are connected by the pre-mRNA. It also elucidated the arrangement of the native spliceosomes within the intact supraspliceosome. Native spliceosomes and supraspliceosomes contain all five spliceosomal U snRNPs together with other splicing factors, and are active in splicing. The structure of the native spliceosome, at a resolution of 20 Å, was determined by cryo-electron microscopy, and a unique spatial arrangement of the spliceosomal U snRNPs within the native spliceosome emerged from in silico studies. The supraspliceosome also harbor components for all pre-mRNA processing activities. Thus the supraspliceosome – the endogenous spliceosome – is a stand-alone complete macromolecular machine capable of performing splicing, alternative splicing, and encompass all nuclear pre-mRNA processing activities that the pre-mRNA has to undergo before it can exit from the nucleus to the cytoplasm to encode for protein. Further high-resolution cryo-electron microscopy studies of the endogenous spliceosome are required to decipher the regulation of alternative splicing, and elucidate the network of processing activities within it.
I will try to sum up the main facts and models detailed in this paper. We should take them with some caution, but they are certainly extremely interesting. a) What we know about the gradual assembly of the spliceosome comes from in vitro studies. b) The authors of this paper have used new techniques to study the spliceosome in its natural context: in the nucleus of cells. c) The results are rather amazing. In vivo, the spliceosome would work in a mega configuration, made of four individual spliceosomes, connected by a pre-mRNA. d) The authors call this structure "supraspliceosome", and each individual spliceosomal unit "native spliceosome". e) The supraspliceosome has a molecular weight of 21 MDa, while each native spliceosome is 4.8 MDa. f) Differently from what is known from in vitro assembly, each unit of the supraspliceosome (native spliceosome) contains all the five U ribonucleoproteins at all times. g) The supraspliceosome is assembled before the splicing. h) Each supraspliceosome includes one complete pre-mRNA, which can be of whatever length or number of introns. Even short pre-mRNAs (like two exons and one intron) are enough to assemble the supraspliceosome. i) The supraspliceosome seems to splice 4 introns at a time, and after that the pre-mRNA is thought to undergo a process of rolling.
The supraspliceosome is a stand-alone macromolecular machine (Fig. 13), composed of four native spliceosomes [29, 30, 37] that are connected by a single pre-mRNA molecule [40], capable of performing splicing of its pre-mRNA [37] – independent of its length or number of introns [6, 31]. ... The supraspliceosome is a multiprocessor machine that can simultaneously splice four introns – not necessarily in a consecutive manner. This configuration enables examination, prior to introns excision, if correct splice junctions will be combined, and allows rearrangement of splice junction combinations to select the appropriate ones, thus ensuring the fidelity of splicing and alternative splicing. The supraspliceosome model predicts that each transcript will be assembled in a tetrameric supraspliceosome. Splicing of a multi-intronic pre-mRNA can be facilitated by the translocation of the pre-mRNA through the complex in a ‘rolling model’ fashion. After processing of four introns the RNA roles in to place a new subset of introns for processing.
j) The supraspliceosome has important and complex regulatory functions:
The supraspliceosome provides a unique and general machine that encompasses the extensive network of interactions and offers coordination and regulation of the different splicing events that a multiintronic pre-mRNA has to undergo. It regulates alternative splicing [39, 40], and harbors a quality control mechanism (reviewed in Ref [31, 41]), as well as all the additional processing activities, such as 5’-end and 3’-end processing, and RNA editing [35, 43]. Furthermore, intronic pre-microRNAs were found in supraspliceosomes and their processing occurs there [44, 45]. Also, a sub-fraction of SNORDs assembled in noncanonical RNPs were found in supraspliceosomes likely playing a role in alternative splicing [46]. Thus, supraspliceosomes harbor components of all pre-mRNA processing activities, thus representing the nuclear pre-mRNA processing machine.
Very interesting, isn't it? :)gpuccio_{December 29, 2017
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ET @240: Exactly. You've got it right. :) Now we're on the same page on this topic. :) Poor gpuccio keeps having that weird "functional complexity" illusion and has come up with a convoluted algorithm to quantify it at the protein level. :) Maybe the breakthrough revelation promised by professor Arthur Hunt will finally persuade our friend to change his mind on the subject? :) PS. BTW, does anybody have a clue when we are going to see professor A. Hunt's next comment posted here? Could that still happen this year? :)Dionisio_{December 29, 2017
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Dionsio@ 238- Of course computer software programs originated from physical processes cuz we originated from physical processes. See? That was easy-peasy. :cool: Seriously THAT is the argument. :roll:ET_{December 29, 2017
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DATCG @230: The authors of the paper you referenced could be uneducated pro-ID Korean folks who don't understand evolution. :) Let's not rely on pro-ID sources to get information from. :)Dionisio_{December 29, 2017
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ET @236: Computer software programs operate on pure physical processes -electronic circuits- therefore they must have originated from physical processes. Is that right?Dionisio_{December 29, 2017
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Michael Behe is a true hero of our era. Love the guy. ET @ 236: Excellent points.Truth Will Set You Free_{December 29, 2017
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Arthur Hunt is very knowledgeable with respect to biology. That said he, like all evolutionists, doesn't have any idea how blind and mindless processes could have produced the spliceosome. As I eluded to earlier all evolutionists for whatever reason think that if something can be described in physical terms, ie physics and chemistry, then clearly materialistic processes produced it. So they always revert to that- "Look we can observe it happening and it is in accordance with physics and chemistry. Case closed." That is their entire argument.ET_{December 29, 2017
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Mung: "You are just a Behe sock-puppet." It's still an honour! :)gpuccio_{December 29, 2017
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gpuccio: I am really happy to be, once again, in perfect accord with what Behe says. You should be. You are just a Behe sock-puppet.Mung_{December 29, 2017
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#231 Gpuccio nice summary.
e) So, the idea would be that trans-splicing, with its discreetly complex multiprotein structures, could be considered an indication of how the spliceosome itself evolved, especially if we accept that spliceosomal introns evolved from group II introns when they invaded the nuclear genome of eukaryotes.
I'm not so sure about the "invasion" story, but willing to listen. Wadda ya know, they reference Koonin. Does he ever sleep? The Intron Invasion! Published 2010
Group II introns have not been found in the nuclear genomes of eukaryotes, but their hypothesized descendants, spliceosomal introns and retrotransposons, are highly abundant in eukaryotes, together comprising more than half of the human genome.
I look forward to Arthur's rejoinder :)DATCG_{December 29, 2017
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DATCG: Fascinating issues indeed. I fully agree with you. I hope we can touch some of them in this discussion, if some time is left! :)gpuccio_{December 29, 2017
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Arthur Hunt (and all interested): The interest for group II introns trans-splicing (or even cis-splicing) in organelles is in part connected to the idea that these scenario could be considered as a model of the evolution of the spliceosome. That idea is more or less as follows: a) Group II introns in bacterial genomes are usually self-splicing. They are relatively autonomous retrotransposon entities, and they use their intrinsic ribozyme activity to self-splice and retro-home. However, even in this simplest scenario the intervention of a protein is necessary, but it is the IEP, a protein encoded directly by the intron itself. b) When group II introns are found in eukaryotic organelles, however, they are usually degenerate, and lack the ORF for the IEP. As a consequence of that, apparently, they need the intervention of other proteins, proteins encoded in the nuclear genome of the eukaryote, to be spliced. c) Such splicing can happen both in cis (the proteins act on one transcript) or in trans (the proteins act on different transcripts, that need to be reconstructed. The second scenario (trans-splicing) is necessary when the original gene has been fragmented, and the individual parts are transcribed separately. Of course, trans-splicing is more complex than cis-splicing, and it seems to involve more complex multi-protein structures. psaA, that we have discussed in detail, is the best model for this kind of scenario. d) Finally, the eukaryotic nuclear genome lacks group II introns, but is repleted with spliceosomal introns. The splicing of these introns requires a very complex structure, our major spliceosome (except for some rarer froms of introns, which are spliced by the moinor spliceosome). e) So, the idea would be that trans-splicing, with its discreetly complex multiprotein structures, could be considered an indication of how the spliceosome itself evolved, especially if we accept that spliceosomal introns evolved from group II introns when they invaded the nuclear genome of eukaryotes. Now, I will try to show what are the similarities between the two scenarios (trans-splicing and spliceosomal splicing), and then what are the differences. Similarities: 1) The main common feature is that the intron is spliced by some complex multi-protein structure, and that the proteins are encoded by the nuclear genome. 2) The second common feature is that the catalytic activity of the splicing is essentially implemented by the RNA component, which retains a similar structure, even if constructed in different ways. I would like to specify that this point is more obvious for the self-splicing of group II introns and spliceosomal splicing. I have not really found details on how the catalysis takes place in trans-splicing, but I imagine that the RNA component may have an important role there too. OK, that's all about similarities, IMO. Now let's go to the: Differences: 1) Specificity vs universality. Trans-splicing, as we have seen, is very specific. The multiprotein structure that effects the reconstruction and splicing of psaA seems to act only on that gene. Moreover, as we have seen, different proteins are neede for each of the two introns. On the other hand, the spliceosome is highly universal. It is very similar in all eukaryotes, and all spliceosomal introns are spliced by the same spliceosome (even if with the great flexibility allowed by the basic structure). 2) Difference in function: Trans-splicing does two different things: it reconstructs the fragmented gene, even participating in the maturation of its parts, like the tscA RNA; and then it effects the splicing of the two introns. The spliceosome does only the splicing, but with all the additional complexity linked to the differences in intron length, sequence and structure, and to alternative splicing and other regulatory processes. Moreover, group II introns are deeply different from spliceosomal introns, even if they could be derived from them. Gorup II introns are rather variants of a similar structure, while spliceosomal introns vary a lot in length, splicing sites, added functions, and so on. Spliceosomal introns also lack any catalytic and retrotransposon activity. That's why the catalytic splicing is "demanded" to the 5 snRNAs in the spliceosome. Finally, if we believe to the meaning of the results presented in post #211, the trans-splicing system seems to serve essentially for splicing, and could not have additional regulatory functions. The additional regulatory functions of the spliceosome-spliceosomal introns system, instead, are beyond any doubt. 3) Difference in conservation: Both the trans splicing complex and the spliceosome are essentially eukaryotic structures. However, while the psa trans-splicing complex is made of proteins that could well be defined as "taxonomically restricted" to that organism, the proteins which make up the spliceosome are, as we have seen, extremely conserved in all eukaryotes. To illustrate this point, I will present here two blasts from two proteins, both of them from Chlamydiosomam reinhardtii: a) Raa1 is probably the main structural protein in the trans-splicing process. I have blasted it against all known sequences in the non redundant protein database of NCBI (practically all know proteins). Here is the result: Protein Raa1 from Chlamydiosomam reinhardtii (Q5K4K9) Length: 2103 AAs Blasted against the whole protein database: Only 4 hits: Volvox carteri f. nagariensis (colonial green alga) 487 bits 318 identities Gonium pectorale (colonial green alga) 352 bits 272 identities Monoraphidium neglectum (single-celled green alga) 171 bits 175 identities Gonium pectorale (colonial green alga) 152 bits 208 identities That's all. A really taxonomically restricted protein. b) Of course, Chlamydiosomam reinhardtii is not only the proud owner of the most studied trans-splicing complex. It also owns a spliceosome, like all eukaryotes. So, I have blasted the Prp8 protein, which is the main structural protein in the U5 subunit of the spliceosome, vs the most "distant" eukaryotic group: vertebrates. Here are the results: Protein Prp8 from Chlamydiosomam reinhardtii (A8HPL0) Length: 2346 AAs Blasted against vertebrates: First 100 hits: Best hit: Patagioenas fasciata monilis (bird) 4040 bits 1889 identities Lowest hit (among the first 100): Clupea harengus (bony fish) 4013 bits 1878 identities A truly universal protein. More than 4000 bits of conservation between green algae and vertebrates. Remember, about 1 billion years. And the amazing thing is the consistency of conservation: only 27 bits of difference between the best and worst of the first 100 hits in vertebrates! With more than 80% identity, this sequence remains practically the same in all contexts! So, these are IMO the main similarities and differences between the two scenarios: trans-splicing and spliceosomal splicing. Everyone can make up his mind about the possible meaning of trans-splicing as a "model" for spliceosome evolution. As for me, I would simply say that they are two very different systems with some similarities. Each of them is highly complex, even if the spliceosome is certainly much more complex. Moreover, the complexity of the splicesome can be approximately and reliably measured through the high conservation of its protein components for extremely long evolutionary times. The complexity of the trans-splicing system, instead, although intuitively obvious, cannot be really assessed, for two important reasons: - we know too little of the detailed structure and biochemical function - the proteins are taxonomically restricted, and therefore we can infer nothing about their functional information from conservation.gpuccio_{December 29, 2017
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Gpuccio, Pardon the pun, but an Intronic Interlude while awaiting Arthur's next comment? What of Introns? Once deemed as "junk" DNA. I'd looked at them in past specifically for potential signs of function and design. Documentation on new functions of Introns is growing. Another angle, but still coordinated with careful splicing and targeting by the Spliceosome. Not to distract from this post. Looking forward to Arthur's follow up. But maybe in future, if not you, someone here @UD could keep track on Intron's functions and possible dual role relationships? Maybe that's stretching it a bit ;-) But something's missing. We're getting the splice events for mRNA, but also producing ciRNAs or other noncoded RNAs. From alternative splicing to Circular Long Noncoded RNAs - ciRNAs. Some reside in the nucleus, others in cytoplasm. Is there added constraint on Spliceosome as a result of Intron functionality? As in wrong splice, wrong dice. How much room for a mistake is there? Circular RNAs are more stable than linear. I suspect more will be found. For anyone who wants something to read, General overview of current progress... Introns: The Functional Benefits of Introns in Genomes(from 2015)
In the present review, we first introduce some studies showing what molecular characteristics of introns cannot be explained by a simple random mutational process that real junk DNAs may have undergone. Subsequently, we summarize the functional characteristics of introns that have been studied providing clues about the adaptive significance of introns in genomes.
more...
Taken together, first introns among all introns within genes have special functional characteristics, indicating that the existence of introns within genes is highly unlikely to be the product of a random process.
Agreed... Addressable subspace must be tightly controlled, otherwise it's all downhill and access is lost. Creating a cascading roll of errors until ending in catastrophic event, or removed by error correction.DATCG_{December 29, 2017
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See the papers referenced in this paper: ... However, the very different mechanism of the RNA editing systems in existence, and their very limited distribution within specific groups of organisms indicate that they are more likely derived traits that evolved later in evolution [65,66]. The sheer complexity of the kRNA editing process, with no obvious selective advantage, led to the proposal that insertion/deletion editing arose via a constructive neutral evolution (CNE) pathway [67]. Indeed, RNA editing in trypanosomes is always mentioned in support of CNE as an example of how seemingly non-advantageous, complex processes can arise [68,69]. ... PLOS NEGLECT TROP D Laura E. Kirby, Donna Koslowsky DOI: 10.1371/journal.pntd.0005989 https://www.researchgate.net/publication/320295574_Mitochondrial_dual-coding_genes_in_Trypanosome_bruceiDionisio_{December 29, 2017
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The Splicing code.

The Prp8 protein.

What are the constraint metrics?

A brief conclusion.

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