Short Straw

Penniless student, silent observer, aspiring scientist, pipette enthusiast.

Splicing Explained III

       

Genetic diseases are familiar to all of us, even if we or a close friend/relative are not directly affected. They are diseases which can be passed on from generation to generation and can affect people for their whole lives. Defects in splicing are responsible for 15-50% of the genetic diseases out there. In this last post on splicing, I want to highlight how such defects can lead to disease using two examples.

Frasier syndrome

Individuals with this syndrome exhibit problems in sex determination and kidney development. They also carry an increased risk of developing tumours in genitourinary regions.

Now if you remember from previous posts, I talked about how alternative splicing can allow different exons to be incorporated or excluded in the final mRNA. This can lead to the production of proteins that have different functions. In a healthy individual without the disease, this is exactly what happens. A particular gene leads to the production of 2 proteins of distinct function. One protein is involved in assisting the production of mRNA from other genes. The other is involved in regulating alternative splicing (we’ll come back to this in a minute). The ratio of these 2 proteins is very important in guiding correct sex determination and kidney development.

In individuals with Frasier syndrome, what happens is that there is a mutation that prevents exons from being included or excluded correctly. Alternative splicing therefore isn’t happening in the appropriate way. The 2 proteins are still made but they are not made in the right quantities. The ratio of the proteins is skewed. If you’re not a scientist, imagine this being a bit like baking cookies. You might need to add both sugar and salt but if you don’t add them in the correct amounts and their ratio is skewed, you’ll either get cookies which are too salty or sickeningly sweet.

So, not only is alternative splicing not happening correctly, one of the proteins which is involved in regulating alternative splicing is being produced at incorrect levels. This protein will affect the splicing of other mRNAs and the correct formation of other proteins.

Myotonic Dystrophy

Individuals with Myotonic Dystrophy (MD) have severe muscle weakness, due to this being a progressive muscle wasting disease, and insulin resistance.

In individuals with MD, a repeat expansion mutation in the DNA causes an abnormal mRNA to be produced. This abnormal mRNA has two effects. Firstly, it sequesters a protein known as MBNL in the nucleus. This means it anchors many copies of this protein and prevents them from doing their normal job which is to regulate splicing. Secondly, it stimulates a protein known as CELF to higher than normal levels. CELF is also involved in regulating splicing. 

During regular development, MBNL activity increases and CELF activity decreases - this is the case in individuals without MD. However, those who do have MD have this balance or ratio disrupted because of the anchoring and stimulating I just described. This disruption in the balance of these proteins that regulate alternative splicing leads to proteins such as the insulin receptor not being produced properly.

What I hope to have done, with these three posts on splicing, is illustrate the importance of splicing. Scientists all over the world are working to understand alternative splicing - how it is regulated, where and by what. Not only because it poses interesting intellectual questions but also because defects in splicing affect many people with genetic diseases and this could potentially be a point of therapeutic intervention in the future.

Science & Film

Have you ever wondered what the first film ever made was? Well, whether you thought about it or not I’m obviously going to tell you. In the late 1800s, a man known as Eadweard Muybridge lined a series of cameras along a race track. Each camera was hooked up to some thread which triggered the shutter shut when a horse ran through it as it raced along the track. Muybridge put all his images together and made the first film which looked like this (if you can’t see it move, click here):

                                

As you’re reading this, I know you’re asking why he chose a horse and why he cared. The reason is that until then, noone actually knew if horses (and other four legged animals that run at such high speeds) had to keep at least one hoof on the ground when galloping. In fact, the understanding of locomotion at the time was quite poor which is reflected by the fact that paintings of horses at the time never showed a horse completely off the ground. This film proved that horses can indeed have all four hoofs in the air. Believe it or not, the study of locomotion (movement) is a field of its own for some physiologists.

What I find interesting about this is that it has been applied on a molecular level, we’re still solving the same problems (albeit on a much smaller scale) in the same way. A group of scientists have taken this concept and used high-speed atomic force microscopy to look at motor protein locomotion in cells. What they see is that myosin 5 (a particular motor protein that they chose to look at) has two “feet” and it uses them one at a time to move forward but will always keep at least one “foot” attached to the surface (actin, in this case).

If you’re super geeky like me, you can watch their video here. 

The above is an image of Coronavirus. Coronavirus takes its name from the latin word ‘corona’, which means crown, because of its characteristic crown-shaped exterior. It is both an enveloped and positive ssRNA virus. In 2002, a novel coronavirus emerged in China that caused SARS (Severe Acute Respiratory syndrome) and subsequently killed approximately 10% of the 8000 people it infected globally. Recently, a new coronavirus has surfaced again, infecting 6 people so far - this time in Saudi Arabia and Qatar. Symptoms share some similarity with SARS and include coughing, fever and breathing difficulties. The World Health Organization are currently working to understand the origin, epidemiology and relatedness of these viruses whilst encouraging increased surveillance.

The above is an image of Coronavirus. Coronavirus takes its name from the latin word ‘corona’, which means crown, because of its characteristic crown-shaped exterior. It is both an enveloped and positive ssRNA virus. In 2002, a novel coronavirus emerged in China that caused SARS (Severe Acute Respiratory syndrome) and subsequently killed approximately 10% of the 8000 people it infected globally. Recently, a new coronavirus has surfaced again, infecting 6 people so far - this time in Saudi Arabia and Qatar. Symptoms share some similarity with SARS and include coughing, fever and breathing difficulties. The World Health Organization are currently working to understand the origin, epidemiology and relatedness of these viruses whilst encouraging increased surveillance.

Splicing Explained II

In my last post, I discussed what splicing is and the basics of what goes on when pre-mRNA is spliced. I highlighted that this is one of the ways cells can produce more proteins than there are genes. In this post, I will put alternative splicing into context by illustrating some of its functions using representative examples. 

             Alternative splicing can create proteins with different functions

              

Let’s approach this by looking at SV40, a virus that infects humans and monkeys. When the virus enters a cell, it loses its protein coat and the DNA it carries is transcribed into pre-mRNA as we saw in the last post. Splicing of this pre-mRNA can produce the ‘large T antigen’ protein or the ‘small t antigen’ protein, 2 different proteins with 2 different functions. You might think they sound the same but their function is hugely different.

The ‘large T antigen’ protein is responsible for causing cancer. It does this by interfering with proteins in the cell (such as p53) which are there to prevent cancer from occurring.

The ‘small t antigen’ on the other hand prevents the cell from ‘committing suicide’. When cells are under significant stress due to infection for example, they may commit suicide (a process known as apoptosis) in order to prevent infection spreading to nearby cells. The small t antigen prevents this from happening and allows infection to spread and continue.

              Alternative splicing can be used to create variability in proteins

               

It might seem slightly counter-intuitive after all I have told you that alternative could produce many different proteins that have the same function. What purpose could this possibly serve? Yet, this is the case. In flies, a single gene known as DSCAM is able to produce more than 38,000 different Ig proteins that all have the same function. If you’re interested, this is done by chosing from 4 sets of mutually exclusive exons but that’s not really as important as what comes next.

Flies have brains, they’re not as complex as human brains, but they’re brains nonetheless and they contain neurons and neural connections. Neurons display these proteins on their membranes but different neurons display different splicing products. Neurons that have the same Ig proteins are repelled from each other and neurons that have different Ig proteins can form connections and pass information along. So all Ig proteins have the same function, they help determine whether neurons form connections or repulsions. However, splicing allows many different types of Ig proteins to be made which help the brain develop properly.

Interestingly, if scientists prevent this variability and allow cells to produce just 500 Ig proteins then regions of the brain do not develop.

                  Alternative splicing can affect where proteins are localized

           

Let’s approach this by thinking of the interior of a cell as one region and the outside of a cell (ie the surrounding environment) as another. Before an infection, B cells of the immune system hang around quite happily with antibody bound to their membrane. A few days after a pathogen is detected, B cells secrete or release antibodies into the environment instead. This is a result of alternative splicing. One type of protein contains a region which allows it to be anchored into the membrane whilst the other doesn’t contain this region and so is secreted from the cell into the environment.

        Alternative splicing can determine if gene expression is ‘ON’ or ‘OFF’

   

Flies are again an excellent example of this. A protein known as Sex-Lethal is involved in the sex fate of a particular fly. This protein is produced in females but not in males because of alternative splicing. It is then responsible for preventing male development in the females by switching “maleness” genes “OFF”. If this protein isn’t there, there is no protein to switch these genes “OFF” and so the fly will be male.

Splicing Explained

Many of you may be familiar with what is considered the central dogma of life, that DNA is first transcribed into RNA which is then translated into a protein. This is the most important process in any living cell. Our DNA contains all the information we need to make a vast set of proteins that are the main workhorses of cells. Proteins make up our metabolic pathways, they act as enzymes, they function as receptors to detect and respond to environmental changes - in short, their uses are far too extensive to be described in a humble little blog post.

                                              

The human genome has roughly 20,000 genes. Each gene is responsible for making a protein. By this logic, we would assume therefore that we can make 20,000 different proteins. Yet, before our body has even been attacked by a pathogen, our cells can likely make 1,000,000,000,000 different antibodies (which are also proteins) to protect us. How is it, therefore, that our genome can produce more proteins than it has genes? How can we account for this disparity?

One of the ways is through alternative splicing, but before we get onto that we should probably understand constitutive splicing first. As we know, a gene must first be transcribed to give RNA (specifically mRNA - messenger RNA). Genes contain introns and exons. Initially, these are both transcribed into pre-mRNA as seen in the diagram below. A mechanism known as ‘constitutive splicing’ then removes the introns and leaves just the exons to give the final mRNA product which goes on to form proteins.

           

Alternative splicing is when certain exons are also removed/excluded whilst others are allowed to be part of the final mRNA. So, using the example above, we could keep the first exon but not the second or third. We could have a situation where we keep the second exon but not the first or third. We could have a situation where we keep the second and third but not the first and so on. Each situation would give rise to a different protein. Therefore this 1 gene could produce a number of different proteins. 

This is a topic I will be doing a series of posts on. It’s a hugely important part of molecular and cellular biology and a failure in splicing is responsible for 15-50%* of human genetic diseases such as Myotonic Dystrophy, Spinal Muscular Atrophy, Frontotemporal dementia with parkinsonism-17, Frazier Syndrome and many more.

(*If you’re wondering about the huge difference in percentages: 15% refers to the genetic diseases that are due to extremely obvious and fundamental errors in splicing, there are also more subtle errors)

Winner of the 2012 Nikon Small World Photomicrography Competition. This is the blood brain barrier of a live zebrafish embryo captured by Dr. Jennifer Peters and Dr. Michael Taylor using confocal microscopy. The blood brain barrier in zebrafish is composed of a single layer of endothelial cells supported by a basement membrane and surrounded by specialized cells such as pericytes, astrocytes and neurons. These cells work together to ensure  the integrity of the blood brain barrier which is responsible for separating the circulating blood from the cerebrospinal fluid. The complex interactions between these cells in zebrafish are a great model to study how cells communicate with each other, it’s also a great model to study embryonic development. Because zebrafish are vertebrates and there is a great deal of conservation between them and higher vertebrates, they are also a great model system in which to discover drugs. This is not just a pretty picture therefore but also quite medically significant! If you’d like to check out the other winners, click here.

Winner of the 2012 Nikon Small World Photomicrography Competition. This is the blood brain barrier of a live zebrafish embryo captured by Dr. Jennifer Peters and Dr. Michael Taylor using confocal microscopy.

The blood brain barrier in zebrafish is composed of a single layer of endothelial cells supported by a basement membrane and surrounded by specialized cells such as pericytes, astrocytes and neurons. These cells work together to ensure  the integrity of the blood brain barrier which is responsible for separating the circulating blood from the cerebrospinal fluid. The complex interactions between these cells in zebrafish are a great model to study how cells communicate with each other, it’s also a great model to study embryonic development. Because zebrafish are vertebrates and there is a great deal of conservation between them and higher vertebrates, they are also a great model system in which to discover drugs. This is not just a pretty picture therefore but also quite medically significant!

If you’d like to check out the other winners, click here.

Yesterday, the Nobel Prize for Medicine was awarded to Sir John Gurdon and Professor Yamanaka. I was surprised at how incredibly emotional this made me, I have believed for a while that these two have done some of the most groundbreaking and interesting research in my lifetime. John Gurdon proved a while ago that all cells contain the same genetic information and that these cells can be reprogrammed to act like specific cell types. Yamanaka proved a few years ago that mature adult cells from a mouse could also be reprogrammed into stem cells and reproduced this with human cells. This has led to breakthrough technology that bypasses the ethical dilemmas of using embryonic stem cells. Scientists can now take “diseased cells” and turn the clock back to watch their development. 

The reason perhaps that this has made me feel all warm and fuzzy inside is that I have always felt Gurdon was a scientist to look up to and aspire to. His experiments are applauded as being highly sophisticated and elegant by his colleagues and anyone in research knows that academia is not a profession filled with back patting and honest compliments. He is an honorary fellow at my college and I have always heard him spoken of with nothing but fondness and admiration. Many academics have said that he is approachable and willing to help all students and colleagues with even the simplest of queries. Perhaps what I admire most of all has to do with a story one of his students told us in our first year. Gurdon wanted to go to university to study science but at school he was actively discouraged from doing so. He was told it was a waste of his time and everyone elses, that his scientific knowledge was poor and he would never achieve anything as a scientist.

I don’t know if he lacked confidence or if he simply didn’t enjoy learning facts. But what I do admire is that Gurdon looks back on this school report when things in the lab don’t go according to plan and he still wonders if that school teacher was right. I admire that someone who has achieved so much, who has an entire institute in Cambridge named after himself, is still able to take a step back and doubt himself. As a person who regularly feels that I don’t deserve to be at the university which I’m at, Gurdon has been one of the few scientists that have inspired me to think that self-doubt can be used in a positive way. 

I once had tea with a brilliant geneticist whose name I won’t reveal here and he said “I’ve never wanted to be special, I just want to be good at what I do”. That’s the impression I get from Gurdon and that’s exactly what I hope for myself too.

This photo is a molecular representation of an antibody. Antibodies are proteins produced by B cells of the immune system. Initially, their function was defined as distinguishing self antigens from non-self antigens. However, this definition was refined to describe their function as distinguishing harmful foreign antigens. Each individual produces millions of antibodies and each of these has two arms (upper part of photo) which are highly specific against a particular type of antigen. This therefore equips the immune system with a defence that is both broad and specific. The arm pictured in the bottom end of this photo is able to attach itself to cells of the immune system, allowing them to come closer to pathogens and triggering them to defend the body. Antibodies can neutralize toxins, enhance phagocytosis of pathogens by opsonization, activate components of the complement system which is involved in defence and much more. In autoimmune diseases, antibodies play a role by recognizing self antigens (eg the fatty insulation around our neurons) or non-harmful, non-self/foreign antigens (eg pollen). This leads to the inappropriate activation of the immune system. This photo is a courtesy of science photo library

This photo is a molecular representation of an antibody. Antibodies are proteins produced by B cells of the immune system. Initially, their function was defined as distinguishing self antigens from non-self antigens. However, this definition was refined to describe their function as distinguishing harmful foreign antigens.

Each individual produces millions of antibodies and each of these has two arms (upper part of photo) which are highly specific against a particular type of antigen. This therefore equips the immune system with a defence that is both broad and specific. The arm pictured in the bottom end of this photo is able to attach itself to cells of the immune system, allowing them to come closer to pathogens and triggering them to defend the body.

Antibodies can neutralize toxins, enhance phagocytosis of pathogens by opsonization, activate components of the complement system which is involved in defence and much more. In autoimmune diseases, antibodies play a role by recognizing self antigens (eg the fatty insulation around our neurons) or non-harmful, non-self/foreign antigens (eg pollen). This leads to the inappropriate activation of the immune system.

This photo is a courtesy of science photo library

When a thing is new, people say: “It is not true”. Later, when its truth becomes obvious, they say: “It is not important.” Finally, when its importance cannot be denied, they say: “Anyway, it is not new.”

-William James

The Other Side of ENCODE

               

Yesterday, I posted a piece explaining what the ENCODE Project was that everyone has been raving about. Today I have woken up to find the criticisms of the way the press release was handled pouring in and I feel that I should reflect this here. Whilst I am still enormously excited about what the ENCODE Project means for science and genetics, many of the criticisms have resonated with me and I feel it is only fair to share those. 

There is a lot of material and it would not be possible for me to cover it all succinctly in one post. I will briefly explain two things that I have been thinking about and I will also link several opinion pieces on these issues and others so that anyone who wants to read more, can.

On the subject that 80% of DNA has a biochemical function:

The issue here, appears to be that people believe this number has been grossly overstated. In my opinion, I think that this partly depends on how you define ‘function’ and whether ‘function’ can be defined by what is ‘interesting’ to you as a scientist. A lot of people are saying that the definition of ‘function’ has been too loose and that a lot of this DNA is serving a very mundane purpose. That may very well be the case but in my opinion, that shouldn’t have any bearing on concluding that it is indeed serving some purpose. Whether or not the number is overstated as a result of estimations is not something I can give an opinion on because I have not read all the papers out there on the topic in enough detail yet.

On the subject that headlines were put ahead of the actual science

It is no secret that science, just like every other industry, is intended to be profitable. It is no secret that magazines, even the most highly respected, strive to be sold out. It has been argued that time was wasted by waiting for the side papers  and main paper to be published at the same time and that scientists could have made better use of the information earlier. It has also been argued that there was intense focus on selling big statements rather than actual science.

                                                                                        ***
I believe criticisms of papers are a healthy and necessary part of the scientific community. This post is not meant to undermine the importance of having the information ENCODE has given us or its significance, it is meant to allow readers to read the criticisms out there. It is meant to highlight that discussion, debate and opinion is important and should always have a place in research.

Here are some pieces that explain things in more detail for those who are interested: