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A quick look at mRNA Splice Variants

-Beadle and Tatum Redux

-Beadle and Tatum Redux

In my microbiology class this past week, we were discussing how prokaryotes and eukaryotes differ in their handling of DNA, RNA and gene regulation. Mostly, we focused on how the presence of the nuclear membrane in eukaryotes separates the processes of transcription and translation and what this results in. Briefly, bacteria are prokaryotic life forms that lack a defined nucleus (among other differences). Because of this, when bacteria transcribe mRNA, it is immediately available for translation – the DNA, RNA polymerase and Ribosome all exist in shared space. Below is a classic image of a strand of DNA(stretching left to right) in E. coli being transcribed into RNA. The RNA molecules extend away from the DNA and appear to travel up or down away from the DNA in this micrograph. Along the length of the RNA, we see dense ribosomes which are busy synthesizing proteins.

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In Eukaryotes, the nucleus encases the DNA, the RNA polymerases and mRNA. mRNA can be completely synthesized and modified in a number of ways before they are exported from the nucleus to the cytoplasm, where ribosomes will translate the message into protein.

One of the modifications of Eukaryotic mRNA we spoke about was splicing. Splicing is a means of snipping segments of non-coding introns out of the mRNA leaving a mature mRNA with a continuous strand of exons. One interesting possibility this enables is the production of alternative sequences made from differential splicing of the immature mRNA. These alternative mRNAs are known as splice variants. At this point, I was asked for an example of a gene that is handled in this way and was caught flat-footed.

Hmm. Perhaps this is something that I’d heard so much about in classes but never in the ‘real world’. I’ll have to look.

One of the first things I found was this discussion of splice variants suggesting that this was not a biologically significant event. i.e., the RNA may be alternatively spliced, but do these splice variants actually result in functional proteins with different properties. The author poses a challenge to find examples of splice variants that are ‘real’. The ensuing discussion is a good one.

What would this looks like?

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Regardless, I found a paper with some good figures that may help students understand how this phenomenon (at least putatively) occurs.  Here’s the best figure presenting a diagram of the different mRNAs created and gels and sequence data indicating that these exist.

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The above Figure shows the presence of distinct RNA species, although that, alone, does not mean that these RNA are ever made into protein. To do that, western blots of protein extracted from various tissues is shown below.

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What is left to find is whether each of these two proteins actually does something. Are both forms required? Are their functions distinguishable?
My quick look through the literature did not uncover any evidence for this last question. If anyone out there knows the literature on this, I would love a push in the right direction. It doesn’t matter what gene we’re looking at, just that it is an example of alternative splicing and that each of the splice variants is actually made and has some identifiable and distinct function.
 
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Posted by on April 4, 2014 in Uncategorized

 

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A quick followup on that lac operon post

Last week I posted a quick link about operons for my micro class to check out before taking their quiz on bacterial gene regulation This post is intended to complement that one. To go back to that post, click here. If there’s one thing to remember about operons it is that bacteria, lacking a nuclear membrane, regulate their genes differently than Eukaryotes. Having a nuclear membrane separates transcription and translation into two distinct compartments allowing for more subtle tweaking of Eukaryotic mRNAs before they are exported for translation.

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Click on this figure to go to a good description of how polycistronic genes work

One thing this does is it makes it very beneficial to package genes with related function closely on the genome and use a single regulatory region to control them all together. They wind up getting packed so closely together that they are actually expressed as a single messenger RNA – known as a polycistronic (meaning ‘many gene’) message.

Upstream of this polycistronic cassette are regulatory elements. One element common to all regulatory elements is the promoter. The promoter consists of several elements which ‘promote’ the binding of an RNA polymerase to the DNA. Additional regulatory elements exist to ensure that this polymerase only transcribes the genes if they are needed. In doing so, the cell conserves energy and components (e.g. Amino Acids) for only necessary processes.

In the case of the paradigm lac operon, lactose is a fuel source, but not as good as glucose. Therefore, enzymes to digest lactose are only needed when lactose is present, but glucose is not. In order to interrogate both conditions, two additional regulatory elements are present.

First, the operator sequence. This sequence binds a repressor protein that physically blocks the polymerase’s path in the absence of lactose. However, if lactose is present, the sugar binds to the repressor, causing a conformational (shape) change that causes the protein to release its grip on the operator sequence.

Second, a catabolite activator protein (CAP) will only bind to the DNA behind the RNA polymerase if cAMP is present. Let’s not get too distracted, other than to say that cAMP levels are high in the ABSENCE of glucose, and low when that sugar is present. When cAMP binds to the CAP protein it can now bind the DNA and do it’s other job: making a nice binding site for the RNA polymerase. Without CAP, the polymerase binds very inefficiently.

Together, the production of lactase enzymes (those that digest lactose) is exquisitely controlled in a way that conserves the most energy.

ImagePs – take a look at this graph and tell me why (not mechanistically, but rationally) the cell does not make lactase enzymes when both glucose and lactose are present.

 
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Posted by on March 15, 2014 in Uncategorized

 

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Codon Usage Bias – Part II

Yesterday I got caught up writing about the way that biologists use codon usage bias to optimize cloned genes for expression. Today, I want to finish this up by discussing two ideas about why codon optimization occurs.

#1: Through some stochastic (random) process, one or more tRNAs corresponding to certain codons was / were amplified through gene duplication resulting in higher efficiency translation when these codon are used. This implies that the efficiency acts as a selection process that re-inforces the once-random preference.

Evidence for this model lies in the observed relationship between tRNA genes and codon usage. Work done at the Institut Pasteur shows that, “By analyzing 102 genomes we showed that as minimal generation times get shorter, the genomes contain more tRNA genes, but fewer anticodon species.”

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Codon usage correlates with gene copy number of corresponding tRNA

#2: Weatheritt and Babu suggest that there are additional codes overlaying that of the DNA->RNA->Protein code. These codes are the result of DNA binding to proteins, RNA looping, or micro-RNA binding that may impose their own restrictions on sequence.

The original paper, by Stergachiset al. of the Stamatoyannopoulos laboratory at the University of Washington, used DNAse footprinting to determines the areas of DNA that were bound by proteins.

Imagine DNA as a long clothesline. In some locations socks hang from the clothesline covering up small areas of the string. DNAse is an enzyme that can chew up open DNA, but is not capable to displacing proteins to chew up the sequences they bind. That is, wherever the clothesline is empty, it is goggled up; wherever a sock hangs, those regions are protected and we can go back to see what they are.

ImageStergachis et al decided to look at these sequences to determine if any of these correlated with the preferred codons for several amino acids that have a number of possible codon alternatives.

What they found actually does account for some of the observed codon bias. In the figure below, taken from their paper, note the difference between the preference for the codon, CTG, if this codon appears in an area where proteins bind to the DNA. This paper does not specifically define the proteins that bind the DNA at any given location, but it is clear that this sequence is vital to two distinct functions.

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CTG codon preference is greater when occurring at a protein-binding site

Because CTG remains a preferred codon even in the absence of protein binding, it is reasonable that both models may be correct. i.e. protein binding may have tipped the balance in favor of certain codons which sets up an environment where multiple tRNA genes for this specific codon, over others coding for the same amino acid, is preferred.

Lastly, I was alerted to the following video blog addressing a different interpretation of the same data:

 
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Posted by on February 10, 2014 in Uncategorized

 

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Codon Usage Bias – Part I

To the molecular biologists:

Optimize ye codons while ye may

For time is a-flying

And this clone you have in R & D today

Tomorrow will be … in manufacturing- and it’s just impossible to change anything at that point, so forget it.

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I”m no rocket surgeon

 Codon Usage Bias – Part I

I read an article yesterday about codon bias that has been stuck in my head ever since. The article, appeared as a ‘Perspectives’ piece in the 13 Dec 2014 issue of Science, with the title, ‘The Hidden Codes That Shape Protein Evolution.’

This article addresses some details not often considered in how DNA directs the synthesis of proteins.

 I spend a lot of time in my classes discussing the basic mechanism by which DNA –>RNA –> Protein, known as the Central Dogma. A lot gets left out of these lectures in order to keep it simple, which sometimes keeps the way I think about the flow of information pretty simplified as well.

Fortunately, this article rattled my cage enough to open my mind to the myriad influences that go into the stuff of life. Here, Weatheritt and Babu, look at how DNA sequences may be under selective pressures independent of just the proteins they encode.

I’ve done a fair amount of molecular biology in my life, including cloning genes and moving them into other organisms for expression as drugs or drug components. One example of this was in a lab where we used live-vectors as immunogens in order to take advantage of the uniquely broad immune response this single-cell pathogen elicits. The immune responses we wanted to trigger / amplify were typically against human tumor proteins or the products of human viruses (e.g. HIV, HPV), however the organism we were using as a vaccine was a bacteria.

As I said above, I usually teach the Central Dogma in a way that omits many of the complications seen in the real world. So, when we look at a codon chart, we see the redundancy (multiple DNA codons make the same amino acid) to illustrate how a change in the DNA sequence can often fail to change the protein sequence at all. These are called ‘silent mutations’.

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The way these codon charts work is by triangulating a position in the middle of the chart using the bases depicted along three of the four edges. For example, the codon AUG is read by locating the ‘A’ on the left margin, the ‘U’ on the top margin, and the ‘G’ on the right margin. The location this identifies is an amino acid called Methionine (abbreviated as met) on this chart.

Notice that if the first two bases in a codon are CU, then it does not matter what the third base is, no matter what, this codon will call for a Leucine (leu).. This means is the sequence of RNA is CUU originally, but mutates to CUC, there will be no change in the protein.

What codon optimization addresses is the fact that different organisms tend to prefer some codons over others, even if they encode the same amino acid. This has been appreciated for many years now so when a molecular biologist takes a protein (e.g. from a human tumor) that they want made by bacteria and they redesign the DNA sequence in a way that codon preferences are maximized in the organism that will express the protein.

This figure examines the percentage of times a gene uses a particular codon to make Leucine. In the bacteria, E. coli, CTG is used nearly 50% of the time. Meanwhile, in the yeast, S. cerevisea, TTA and TTG are preferred.

 (Note that T in DNA = U in RNA)

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So, what does this mean? Consider a simplified example…

I want to clone this protein from a yeast and grow it up in bacteria:

Met – Leu – Leu   [stop]

ATG- TTA – TTG – TAG

 We would take this DNA from the yeast and then modify the sequence by changing the two Leucine codons into the preferred sequence in bacteria (CTG):

Met – Leu – Leu   [stop]

ATG- CTG – CTG – TAG

The result should be a sequence of DNA that the bacteria will be able to optimally translate into protein. 

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This has worked out to be much longer and more technical than I intended – and I haven’t even addressed the new ideas brought up in the Science article.

Therefore, I’m going to stop here and continue tomorrow with part II

 
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Posted by on February 9, 2014 in Uncategorized

 

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eteRNA RNA folding game

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Proper Folding Achieves Lowest Energy Conformation

My general biology students were asked yesterday to check out eteRNA, an interesting online game designed around problems of RNA folding. RNA is a fascinating molecule for a number of reasons.

  • What we might think of immediately, mRNA as an information carrying molecule, is just one of its many jobs.
  • In addition to this, RNA serves as a delivery molecule in the form of tRNA. These molecules are capable of both ‘reading’ the mRNA message, through codon:anti-codon interactions, and they also recruit and deliver Amino Acids that correspond to the codon in question. 
  • RNA also functions as ribozymes, enzymes comprised entirely, or in part, of RNA. An example of this is the ribosome that is mostly rRNA with only a small protein contribution.

EteRNA explores the plasticity of RNA function by demonstrating the capacity of this molecule to fold into a variety of shapes. As I frequently remind my class, FORM DICTATES FUNCTION. This is true of all things in biology (and perhaps beyond). When a molecule is formed correctly, it carries out its function appropriately. When that form is altered due to mutation, misfiling or other denaturing processes, the function is also altered. This may be for better or worse, but mostly for worse.  Depending on this function, the ‘fitness’ of the cell for survival / reproduction may be affected leading to selection for or against this cell.

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Eterna

I set up a group titled: FortScott_Treml that I invite my students (or anyone else) to join. For my students, anyone who completes the the tutorials will receive 5 extra credit points. Anyone who earns a puzzle master badge will earn another 5.

 
 
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Posted by on November 13, 2013 in Uncategorized

 

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RNA World and the Origin of Life

In 2011  Martin Hanczyc delivered a TED talk in London on the topic of the origin of life, “The Line Between Life and Not-Life” that discussed some of his work with proto-cells. I participated in some online commenting on the TED page including a conversation about the origin of genetic material.

I wanted to point to the talk itself and include some of the posts below.

ImageFrom Ted Mozer III: ” Two questions about life and the origin of same on earth:
Is all know life on earth related and DNA (or even RNA) based?
If life was created on earth (and not from a seed that either arrived via an comet or the like or from an alien visit), why is the creation process a not a contining process. Did the creation process occur and then stop once life awoke? If so, why??”

My Reply: “You’re asking a very good question, Ted.
Think of it this way, imagine that life first originated by self-replicating molecules (probably RNAs) that found a nice safe home in some protocells that were floating around in the neighborhood – it doesn’t matter if this is absolutely true or not, just consider the abstract idea. The ‘food’ that these cells need is more RNA and cell membrane material, right? So, the things that these cells will ‘eat’ are exactly the same stuff that they, themselves, once were. If our new cells are successful, they are probably gobbling up all the other pro to-life material around them.
This does not mean that life could not have happened more than once, but if it’s a rare enough event, then the first things to get there are going to probably stay at the top of the heap.

It would be really cool to find organisms that use different genetic material – this would support multiple origin events, but so far, the universality of DNA argues that it was a one-off thing.”

A Comment by an unknown person: “Self replicating RNA? RNA and its components are difficult to synthesize in a laboratory under the best of conditions, much less out in a primordial mud puddle. This is highly unlikely. Yes, this was a miraculous “one-off thing.””

My Reply: “Yes, I agree, it is difficult to conceive of RNA as a self-replicating genetic material that also acts as an enzyme. Although RNA does currently act as genetic material, this role is restricted to viruses while DNA plays the major role of genetic material in all other organisms (including some viruses). Also, much of the enzymatic work in biological systems is currently carried out by enzyme proteins. However, there are still some RNA enzymes (ribozymes) extant, one of note is the ribosome – a protein / ribozyme complex with deep phylogenetic roots.

The idea of an RNA world as life’s origin has been around for some time, with suggestions of such an origin being proposed by Francis Crick, Alexander Rich and Harold White (among others) in the 1960s and 1970s.

Over the years, data has emerged supporting such a possibility including:

“The system, created by Gerald Joyce and Tracey Lincoln at the Scripps research institute in La Jolla, California, involves a cross-replicating pair of ribozymes (RNA enzymes), each about 70 nucleotides long, which catalyse each other’s synthesis.  So the ‘left’ ribozyme templates the synthesis of the ‘right’, which in turn templates the ‘left’ and so on, building each other via Watson-Crick base pairing. “

discussed in “Chemists edge closer to recreating early life”, Royal Society of Chemistry 2009.

also,

“Clemens Richert and colleagues at the University of Karlsruhe have now shown that, without the use of enzymes, an RNA strand bound to a longer template strand of RNA can grow more than one order of magnitude faster than previously believed. This growth occurs in single nucleotide steps according to the base pairing rules of Watson and Crick.”

-From “Accelerating non-enzymatic RNA replication“, Royal Society of Chemistry 2005.

However, support is not proof. There will never be proof of what actually happened, but, then again, I might just be a brain floating in a jar somewhere…

 
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Posted by on October 13, 2013 in Uncategorized

 

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“Life is nothing but an electron looking for a place to rest”

ImagePhysics -> Chemistry -> Biology

The Smithsonian Magazine has an article this week proposing that we consider Mars as the origin of Terrestrial Life. This notion stems from Steven Benner’s Four Paradoxes: The Tar Paradox, The Water Paradox, The Single Biopolymer Paradox and The Probability Paradox. Each of these is described in the abstract of his work, and do add up to a possible alternative for life’s origin. However, as compelling as his arguments may be, the origin of life will always be a mystery veiled in time. Even if we were to find evidence of life on Mars that is very much like that on Earth, it would be difficult to say whether Terrestrial life was the origin of Martian life, or vice versa.

Another problem I have with tracing the origins of life off-planet is that it does not solve anything, but merely relocates the source. So it’s not that I feel that Benner’s work is uninteresting or unworthy of consideration, but presently, Ockham’s razor precludes Imageseriously considering extra-terrestrial origins without a good deal more hard evidence. Further,  relocating the source or life’s origin does little to change how we think about  origins. Regardless or where life started, it is still highly probably that it began with RNA, a unique molecule in that even today it serves dual roles as an information-carrying molecule and a structural one that often has enzymatic function. And, that the addition of the more stable , DNA molecule as the primary source of information happened later – as adding protein synthesis also did for providing an alternative structural / functional molecule. 

Evolution of the Central Dogma?

                                                              RNA

                        DNA -> RNA                                                  RNA -> Protein

                                                  DNA -> RNA -> Protein

 
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Posted by on August 31, 2013 in Uncategorized

 

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