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Author Topic:   MicroRNA as Evidence for Evolution
Taq
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Message 1 of 7 (791742)
09-20-2016 4:29 PM


Appropriate forum would be "Biological Evolution".

For this topic, I would like to discuss the viability of microRNA's (miRNA) as an accessible piece of evidence for evolution.

miRNAs has several attractive features for these types of discussions.

1. Short sequence (~90 nucleotides).
2. High conservation between species.
3. Accessible online tools for viewing miRNA sequences.

A gene can be tens of thousands of bases long with multiple introns and exons. miRNAs can be viewed in a large font in a single line. This alone makes them more accessible to the average person.

One of the difficulties I have found when discussing these topics is aligning DNA sequences for specific genes across specific species. It is doable, but sometimes the effort doesn't seem worth it for these types of discussions. However, microRNAviewer does all of the heavy lifting when it comes to miRNAs. If you look at the large table at the link above, the left hand column lists the specific miRNA. The row across the top gives the three letter code for different species (e.g. hsa=human, ggo=gorilla, mmu=mouse).

If you click on mir-34 in the left column, it brings you to this page which shows you the multiple variants of mir-34, and the known sequences for that mir-34 variant in different species.
If you click on mir-34a, you reach this page, which is the real gist of this thread. Again, you have the three letter codes for each species on the left, and the sequence for mir-34a in the middle of the page. At first, it looks like a bit of a jumble towards the top of the page, with multiple colors, dashes, and letters. However, it is all summarized quite nicely at the bottom of the page, where you see this ASCII figure:

                       **************************           * * ***********    * ****                            
**** ************************** * * * *************** ******* *
** **** ************************** * * * * ************************ ****
***** ** **** **************************** * * * ************************ ****
***** ******** **************************** * * * ************************ ****
***** ******** **************************** * * ** ************************ ****
******************************************* * * **************************** **** *
******************************************** * * ********************************* * *
******************************************** * * ********************************* * * ***
******************************************** * ** ************************************** *** *
******************************************** * ** ************************************** *** **
** ******************************************** **** * ************************************** *** ***
** ******************************************** ****** ************************************** *** ***
*** ******************************************** ****** ****************************************** ***
************************************************ ****** ***********************************************
************************************************* ****** ***********************************************
********************************************************************************************************
********************************************************************************************************
******************************************************************************************************** *
******************************************************************************************************** **
******************************************************************************************************** **
******************************************************************************************************** ****
******************************************************************************************************** *****
**************************************************************************************************************
**************************************************************************************************************
**************************************************************************************************************
**************************************************************************************************************
***************************************************************************************************************
***************************************************************************************************************
***************************************************************************************************************
***************************************************************************************************************
***************************************************************************************************************

What is this showing? The bottom row of *'s is the human sequence. Each line above it is another species, with the species more closely related to humans towards the bottom, and very distantly related species towards the top. Each * in the row indicates a match to the human sequence. Each gap indicates a difference between the two species.

What you see is that for closely related species the sequence is identical, and you only see differences when you start to look at more distantly related species. What is of much greater interest is where in the sequence you see the differences in those more distantly related species, which will be discussed in later posts.

In further posts we can discuss what miRNAs do, their maturation process, and why only evolution can explain the pattern for both the similarities and differences between miRNAs found in different species.


  
AdminAsgara
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Message 2 of 7 (791744)
09-20-2016 4:35 PM


Thread Copied from Proposed New Topics Forum
Thread copied here from the MicroRNA as Evidence for Evolution thread in the Proposed New Topics forum.
    
Taq
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Message 3 of 7 (791745)
09-20-2016 5:14 PM


miRNA secondary structure
I am going to try and break this down into bite sized chunks instead of putting everything into one huge post.

The first topic is RNA secondary structure. Primary structure is the sequence itself. Secondary structure is the three dimensional structure that RNA can fold into due to interactions between the bases on the RNA molecule.

An apt anaology is tying a bow with a ribbon. Certain parts of the ribbon are stuck together while other parts are not, producing knots and loops. RNA can do the same thing. Bases that stick together form the knots (called "stems" in biological parlance), and bases that do not readily stick together form the loops. Bases that like to stick together are called complementary bases. A and U (no T in RNA) like to stick to one another, and C and G like to stick together. For each base, there is just 1 complementary base and three non-complementary bases, which will become important later on. This is due to the number of hydrogen bonds available on each nucleotide, and how those available hydrogen bonds line up with one another.

miRNAs like to form secondary structures called stem-loops. It resembles a lollipop:

The 5' indicates the start of the RNA molecule, and the 3' indicates the end of the RNA molecule. As you can see, complementary bases stick to one another to form the stem, and the noncomplementary bases form the loop structure in the middle of the molecule.

The first clue to understanding the bigger picture is the placement of the loop structure in the overall sequence. That loop structure is found in the middle of the molecule. Where do you see the most differences between homologous miRNA sequences in distantly related species? In the middle of the miRNA molecule. Where do you see the least number of differences? In the stem portion of the miRNA molecule.

For any single nucleotide, there is just one base that is complementary. There are 3 bases that are noncomplementary. Loops allow for more neutral mutations because a noncomplementary base can be swapped out for another, kind of like a synonymous mutation. Not so for complementary bases since there are no synonymous complementary bases.

In future posts, we can discuss the ultimate function of miRNAs and their maturation process.


Replies to this message:
 Message 6 by caffeine, posted 09-21-2016 12:56 PM Taq has responded

  
Taq
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Posts: 6014
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Message 4 of 7 (791763)
09-21-2016 10:27 AM


miRNA Maturation
The primary-microRNA described in the previous post is just the infant stage for a miRNA. There are several more steps before you arrive at a functional miRNA, and it has implications with respect to sequence conservation.

Here is a figure from Nature outlining the process of miRNA maturation:

There are several steps in the maturation process.
1. The primary-miRNA is the RNA molecule transcribed by RNA polymerase from the DNA genome.

2. The pre-miRNA is cut out from the longer primary=miRNA molecule.

3. The stem-loop secondary structure of the pre-miRNA is recognized by transport proteins that line the nucleus of the cell, and the pre-miRNA is transported to the cytoplasm of the cell.

4. Dicer proteins recognize the stem-loop structure of the pre-miRNA in the cytoplasm and clip off the loop structure, producing two separate miRNA molecules bound to one another by complementary bases.

5. One of the miRNA molecules is degraded, leaving a functional and mature miRNA. There are also occasions where both miRNA molecules are allowed to survive and both have function.

The important thing to note is that several features of the primary-miRNA do not survive during the maturation process, and are not a part of the functional miRNA. Those structures are the stem features that are clipped off when the pre-miRNA is released from the larger RNA transcript and the loop structure that is removed by the Dicer proteins once the pre-miRNA is transported to the cytoplasm. For the most part, all you need is a loop of non-complementary bases in order to get transport into the cytoplasm and push the pre-miRNA down the maturation pathway. Any old loop will do. This allows for much more freedom in the sequence making up the loop structure.

Edited by Taq, : No reason given.


  
Taq
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Message 5 of 7 (791765)
09-21-2016 10:52 AM


miRNA Function
The question that you may ask, if you have survived the thread so far, is what in the heck do miRNAs do?

The answer is they regulate the production of proteins. miRNA are a form of post-transcriptional mRNA regulation, to use the sciencey term. If you are familiar with the Central Dogma of molecular biology then you know that the pathway for genes is DNA to RNA to proteins. RNA is transcribed from DNA, and proteins are translated from messenger RNA (mRNA). The bit you may not be aware of is the process of translation itself.

In order for translation to occur there needs to be an interaction between the messenger RNA and several different proteins including the ribosomes that translate the messenger RNA and what are called inititation factors. The whole complex forms a hoop structure (not to be confused with the stem-loop structure from before). Here is yet another diagram showing this structure:

The ribbon running through the structure is the messenger RNA. The proteins that bring the two ends of the messenger RNA together are the initiation factors, and they have to be there in order for the ribosome to bind and begin translating the messenger RNA into protein.

So where does miRNA fit into all of this. The miRNA, with the help of other proteins, binds to the end of the messenger RNA and prevents the initiation factors from joining the two ends of the messenger RNA together, thereby preventing translation of the protein. This binding is determined, once again, by complementary bases between the miRNA molecule and DNA. If the sequence of the miRNA matches up to sequence at the end of the messenger RNA molecule (called the 3'-UTR or 3' untranslated region). In addition, bound miRNAs can also mark the messenger RNA for degradation. This is yet another level of post-transcriptional regulation.

When we talk about functional miRNAs we often mention a seed region. This is the region of the mature RNA that has the complementary bases that match up to the end of the messenger RNAs. As it turns out, a single miRNA can bind to many different messenger RNAs from many different genes, allowing them to control the production of numerous proteins. There are also online tools that can search for both theoretical targets and experimentally proven messenger RNA targets for miRNAs. Some miRNAs can bind to and regulate hundreds of messenger RNAs. For this reason, one small change in the seed region of the miRNA can result in a change in regulation for many genes. This means that changes in the seed region of the miRNA can be quite deleterious which puts miRNA under strong negative selection.

This relationship between messenger RNA and miRNA is not symmetric. The end of the messenger RNA can have several sites where the same miRNA can bind which means one site can mutate while the others are still functional. In addition, mutations in the messenger RNA can prevent the binding of an miRNA, but this only results in a change in regulation for that messenger RNA, not for a whole suite of hundreds of different messenger RNA. Mutations in the messenger RNA can also result in different miRNAs regulating translation of that protein.

This is why you see such strong conservation of the seed regions between species for miRNAs. If you go back to the first post, you will notice that one ~20-25 base pair region on the left hand side of the chart is strongly conserved between very divergent species. This is the seed region. This is the functional part of the miRNA molecule.

This is the type of divergence we would expect to see from evolution. The more time since common ancestry the more differences we should see in genomic regions that can change without deleteriously affecting fitness. We should also see conservation of sequence that is strongly constrained. That is, conservation of sequence that can't change without producing deleterious effects.

So the question for the ID/creationist folks is why change the rest of the molecule? Why not use the same sequence in the loop structure of the pre-miRNA? Why change the sequence in the loop structure so that it matches evolutionary distance? Such changes are not needed since any loop structure will do. We are often told that a designer will copy previous designs, so why not copy the loop structure?

ID/creationism can not explain the chart found in the first post. Evolution can. This is why I believe miRNA is a good source of evidence for evolution, and disproves ID/creationism.

Let me know what you guys think. Is it too complicated? Did I do a poor job of communicating the information? Has this been helpful at all?

If you have made it this far into the thread, thanks for taking the time and effort!

Edited by Taq, : No reason given.

Edited by Taq, : No reason given.

Edited by Taq, : No reason given.


  
caffeine
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Message 6 of 7 (791774)
09-21-2016 12:56 PM
Reply to: Message 3 by Taq
09-20-2016 5:14 PM


Re: miRNA secondary structure
The first clue to understanding the bigger picture is the placement of the loop structure in the overall sequence. That loop structure is found in the middle of the molecule. Where do you see the most differences between homologous miRNA sequences in distantly related species? In the middle of the miRNA molecule. Where do you see the least number of differences? In the stem portion of the miRNA molecule.

Unless I'm misunderstanding something, this seems to be flatly contradicted by the picture in your previous post. It shows that the ends of the molecule are more prone to vary than the middle. The middle is identical in all primates and rodents, while the ends start to vary once we hit monkeys.

I think I am a bit lost somewhere, though, as the ASCII picture doesn't seem to match the listed sequences. Based on the stars, the macaque sequence varies from the apes only in its final base, but the sequence written above also shows two As where apes have Gs (in the 96th and 100th position, counting from the left). What am I missing here?


This message is a reply to:
 Message 3 by Taq, posted 09-20-2016 5:14 PM Taq has responded

Replies to this message:
 Message 7 by Taq, posted 09-23-2016 10:20 AM caffeine has acknowledged this reply

  
Taq
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Posts: 6014
Joined: 03-06-2009
Member Rating: 3.6


Message 7 of 7 (791850)
09-23-2016 10:20 AM
Reply to: Message 6 by caffeine
09-21-2016 12:56 PM


Re: miRNA secondary structure
caffeine writes:

Unless I'm misunderstanding something, this seems to be flatly contradicted by the picture in your previous post. It shows that the ends of the molecule are more prone to vary than the middle. The middle is identical in all primates and rodents, while the ends start to vary once we hit monkeys.

This may be a matter of viewpoint. Since I work in the field of molecular biology I tend to view these molecules with reference to the sequence, not their physical secondary structure. The bases that make up the loop structure are in the middle of the pre-miRNA sequence, while they are at the end of the physical secondary structure that they fold into.

I think I am a bit lost somewhere, though, as the ASCII picture doesn't seem to match the listed sequences. Based on the stars, the macaque sequence varies from the apes only in its final base, but the sequence written above also shows two As where apes have Gs (in the 96th and 100th position, counting from the left). What am I missing here?

You are right. I will have to look into that. However, if you look at the red bases you can still clearly see that the mismatches occur outside of the seed region (which is in blue).

Edited by Taq, : No reason given.


This message is a reply to:
 Message 6 by caffeine, posted 09-21-2016 12:56 PM caffeine has acknowledged this reply

  
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