A Splicing Primer

The central dogma of molecular biology DNA-> RNA-> Protein shows the direction of flow of information of how the cells use the information stored in our DNA to make the necessary proteins. But the situation in most eukaryotes is a little more complex than that simple statement. In most eukaryotes, a gene sequence in a DNA is interrupted by non- coding information. Hence to make a protein, a cell first has to transcribe the gene (make a RNA copy of the gene, called pre-mRNA) and then modify the pre-mRNA by removing the non-coding sequence (intron) and joining the coding sequences (exons) together. The modified mRNA is then exported from the nucleus (where it was made) to the cytoplasm where the ribosome uses it as a template to make the protein. In simple English, the gene for making a proteinA looks like this "HEREabhjhdyfrhUSEndcbldfhdfmMEd ldshhglgmcFORdbfhdflhfnmc PROTEIN A". The task of the cells is to remove the gibberish and make a readable text out of the given instruction - HERE USE ME FOR PROTEINA. The cells then send this information to the ribosome (the protein factory) to make the protein.

Pre-mRNA splicing is the process in which the intronic sequences are removed within a large RNA-protein complex called spliceosome.

Why is splicing important? A spliceosme can remove the non-coding introns present in a given transcript varying combination in response to cellular cues, a process called alternative splicing. The recent completion of a draft of the human genome indicated that more than 59% of the human genes seem to be alternatively spliced (Hastings and Krainer,2001) and thus we can have more complexity (make a larger number of proteins) without increasing the number of genes present. For eg, the Dscam gene in flies has 38,000 alternatively spliced isoforms from four variable exon clusters!
More importantly, it is estimated that aberrant splicing causes about 15% of genetic diseases in humans (Philips and Cooper,2000). Thus, the spliceosome plays a critical role in generating the right template for making a protein and any abnormality in this process would be deleterious to the organism.

What do we know about this process? From genetic and biochemical experiments in the humble budding yeast, scientist have been able to understand how this process occurs. Because both the mechanism of splicing and the splicing machinery are highly conserved throughout eukaryotes, knowledge of yeast splicing gives us insights into the basic process in humans.

The spliceosome is the largest structure in the cell and is composed of five small nuclear RNAs ( called U1, U2, U4, U5 and U6 snRNAs) and over 100 different proteins (Stevens and Abelson , 2002). Under standard in vitro (i.e. in a test tube) assay conditions, the spliceosome assembles in a step wise manner through the addition of the U1 -> U2-> U4/U6.U5 snRNP particles (the small nuclear RNA along with its associated proteins, represented by a colored blob in the picture) on the pre-mRNA (See Figure). This assembly is an expensive process for the cell as each step consumes energy. But it also allows the apparatus to check each step and hence allows for a greater control over the overall process. Remember, a single mistake here would result in a protein that either does not function or functions abnormally. That to a cell would be hazardous and hence the cells err on the side of caution. After the assembly of the spliceosome, it undergoes structural rearrangements, resulting in the loss of U1 and U4 snRNAs, to become catalytically active (Brow D. A, 2002). Then, it proceeds to remove the intron by two transesterification reactions.

The resultant message is released from the spliceosome along with the intron. The spliced RNA is exported to the cytoplasm for translation into the protein and the intron degraded by enzymes in the cell. The spliceosome is disassembled and the components (proteins and the snRNAs) recycled for another round of splicing.

Though much is known about the overall process, there is no insights into what triggers the activation. What informs the spliceosome that everything is set in place and hence go ahead and splice? How does the cell control the ATP driven helicases that remodel the spliceosome at each step? Or what cues the cell about abnormal spliceosome and how does it take a stalled spliceosome apart?

Next time I will try and address the role splicing plays in Humans. How does a cell choose which exon to keep? How do DNA elements present in the gene (ISEs) affect choice of exon? Does the rate at which the transcript is made affect exon choice? So keep your eyes out for Splicing -part deux.

References -
Brow D. A, Annu Rev Genet., 2002, Jun 11; 36:333-60.
Hastings and Krainer, Curr Opin Cell Biol., 2001, Jun; 13(3):302-9
Philips and Cooper, Cell Mol Life Sci., 2000, Feb;57(2):235-49
Stevens and Abelson , Methods Enzymol. 2002;351:200-20.
Check this Animation

A Beautiful Mind

"Imagine if you'd suddenly learnt that the people, the places, the moments most important to you were not gone, not dead, but worse- had never been. What kind of hell would that be?". - A Beautiful Mind, 2001.

I saw the movie for the second time last night and it got me thinking about the complex disorder that is schizophrenia, and the intense effects it has on an individual, making him lose the distinction between real and imaginary. So what is it that makes a person harbour irrational thoughts and so convinced about the his false fears? I tried to poke around the literature to try to understand how much of the organic basis for this disorder is understood. There is the genetic component- the heritable nature of this disorder has been well documented over the years. Mutations in genes that are involved in brain function can be inherited, causing offspring of schizophrenics to be that much more at risk of developing the disorder. The environment plays an equal role, stress and psychological trauma are known to have a causal or triggering effect in schizophrenia, translating genetic predisposition to development of the disorder.

The neuropathology of the disease itself is closely linked to the above described factors. Bad genes, as well as early trauma to the brain, prenatal exposure to infections and psychological trauma result in brain abnormalities that cause cognitive defects and result in the disorder. There are two aspects to understanding how impaired brain function leads to this condition. Firstly the anatomic location of neural systems that are disrupted govern the types of symptoms exhibited by a patient. Various regions of the brain are involved in different functions such as processing impulses, perceiving thoughts and producing a reaction to a stimulus. The distortion in reality observed in schizophrenics is attributed to one region of the brain, thought disorganisation involves malfunction of a different circuit, while a decline in perceptive and physical responses are traced to malfunction in yet another circuit.

Secondly, brain chemistry- in terms of fluctuations in neurotransmitters (the chemicals that transmit signals in the brain cells) control the duration of above mentioned symptoms, to add another layer of complexity to this intricate orchestration. The sum effect of all of this is disorganised thinking, delusional and paranoid thought processes and auditory hallucinations that manifest as schizophrenia.

Dopamine, glutamate and NMDA are some of the neurotransmitters that have been implicated in schizophrenia. The "Dopamine hypothesis" is particularly famous, as it was one of the first major biological causes that could be attributed to schizophrenia. However, it is now thought to be an oversimplification at understanding the disorder, since there are other factors that play a role. Nevertheless, I will discuss the hypothesis because it provides atleast some insight into the process, and is quite fascinating.

Dopamine is a neurotransmitter, and in one of it's functions it is associated with the "pleasure system" of the brain, providing feelings of enjoyment and motivating a person proactively to perform certain activities. Essentially, it mediates the conversion of an outside stimulus from being a "cold" or neutral bit of sensory information into an "attractive" or an "aversive" entity.

For example- normally an external stimulus such as a bright red sports car zipping past a pedestrian might result in a surge of dopamine to cause an appropriate reaction- like the pedestrian turning his head to look at the car. However, the reaction elicited also depends upon the the pedestrian's predispositions and experiences. A race-car enthusiast may turn to look, while a person not interested in sports cars will not exhibit any reaction. In any case, dopamine here mediates a contextually relevant reaction.

In the 1970s, it was discovered that drugs that block dopamine function reduced psychotic symptoms. Further studies led to the hypotheses that dysregulated dopamine transmission causes an abnormal release of dopamine, so that what would have been a normally neutral stimulus results in firing up of neurones and causes aberrant reactions to external objects or their internal representations. Remember Nash in the movie reacting to something as simple as his wife turning on the light by saying "Why did you turn on the lights? Why would you do that? Why?" ?

In this stage, the patient develops a sense of anxiety and confusion, and an intense need to make sense of the new "realities" being experienced. Any and every normal occurrence can produce an exaggerated response in his mind, and he keeps looking for meanings and explanations to calm himself down. As he forms delusions in his mind to explain the occurrences, he experiences a feeling of relief and reduced perplexity. These delusions then persist, even after the stimulus is taken away, eventually taking on a life of their own. Hallucinations arise from similar aberrant thought processes, as the patient conceives an incorrect internal image of a thought or a memory that is percieved and reinforced with such intensity as though it were real.

This is, like I said earlier, just one aspect of cause and development of psychoses but enough to give us a peek into the on-goings in the brain of a schizophrenic. Imbalances in other neurotransmitters and pathways have different ways of interfering with normal thought process and causing psychological disturbances. Given the limited knowledge and understanding of this disorder, how is it brought under control?

Anti-psychotics are useful in the treatment of psychoses, because, in one way, they dampen the effect of the excessive dopamine (in this example) and thus restore a chemical balance that calms the patient. However, they do not change the underlying thought process- all they can do is prevent neutral stimuli from producing abnormal reactions, and quenching aberrant reactions produced initially. Thus, patients are able to "ignore" or control their reactions to stimuli, but are not entirely free of the delusional thoughts that have already formed. This underscores the importance of staying on the drugs as long as is necessary, and also protecting the patient from high stress environments that can cause a resurgence of symptoms. Modern drugs are now being developed to limit side-effects in patients. Imaging technologies have improved to better visualise brain abnormalities associated with schizophrenia. With the availability of genome sequences and better tools, more genes are being discovered that may play a role in the disorder. Emerging tools in pharmacogenomics can make the best of these discoveries to improve treatment. Social acceptance and sensitivity towards the ailment is also needed , to create a support system that does not stigmatise patients.

John Nash's story is a very encouraging one in the face of this complex disorder. His story shows that one can be successful in bringing the disorder under control to a large extent. Eventually, Nash learns to ignore his irrational fears and focus on his passion. The same brain that gave rise to abnormal thought processes also contributed to his Nobel-prize-winning work on the game theory. Indeed, the mind is a beautiful thing!

References: 1) Wikipedia
2) Schizophrenia: challenging the orthodox McDonald et al
3) Schizophrenia in a molecular age. Carol A Tamminga

How does a digital camera work?

In the previous post, I had talked about how a digital photograph is stored in the computer. In this post, I will talk about how a digital photograph senses the photograph talking only about the essential components (using description for RGB colors). A modern digital camera has far more advances than the simplistic picture explained here.

A digital camera has a number of lenses which focus light onto chips that are sensitive to incoming light. In the market, there are two types of image sensors - charge-coupled device (CCD), and the Complementary Metal Oxide Semiconductor (CMOS). CCDs [1] are far more popular than CMOS chips because they are considered to be affected by noise to a lesser extent (and I will use CCDs to explain how a digital camera works). The role of this chip is to sense the light that comes in and convert the light energy to an electric signal that is amplified and then digitized and finally processed.

How a CCD works? Photoelectric effect [2] is the property by which some metals emit electrons when light shines on them. The CCD in the digital camera is a silicon chip that is covered with a grid of small electrodes called photosites. One photosite corresponds to each pixel.

Before a photo is taken, the camera charges the surface of each photosite with electrons. When light strikes a particular side of the photosite, the metal at that site releases some electrons, which travel to the opposite end of the site (forming what is commonly called the capacitor). The larger the intensity of the light that falls on it, the larger the number of electrons that are released, and hence larger the voltage that develops across the photosite. The voltage is then converted to a number using an analog-to-digital converter that corresponds to the intensity of the light that falls on that site. This takes care of the intensity, but we have not discussed about how the photosite knows the color of the light.

As discussed earlier, the color of a pixel is formed by mixing red, green, and blue colors (RGB). So all the light does not hit each photosite, but rather, there is a filter placed on top of the photosite that only lets red, green, or blue color through. Hence, depending on what color is through, each photosite only measures the intensity of the red, green, or blue color that falls on it, and no other color. After this, to measure the intensity of green and blue colors on a site with a red filter, an interpolation algorithm (a process called demosaicing) is used that approximates the intensity of the blue and green light on that site using the intensity of these colors in the neighboring sites.

Lastly, as green is in the center of the spectrum in the visible light (VIBGYOR), our eye is better at picking up different shades of green, and hence, there are a larger number of photosites that sense green light than blue or red. The Bayer pattern shown below is the most common arrangement of photosites in a single array CCD chip.



The other end of expensive digital cameras (read 10's of thousands of dollars) have multiple arrays and avoid the interpolation step. So the incoming light could be split into three copies and then passed through three separate filters and three different arrays and sensed separately to make the final picture by merging these readings together.

There are more complications that arise even in the simple camera, but maybe another post to deal with them (but no promises as I got to do some research before I can post myself).

[1] CCDs were invented by George Smith and Willard Boyle at the Bell Labs.
[2] Albert Einstein won the Nobel prize in 1921 for the quantum explanation of photoelectric effect.

Source:
The source for most of this stuff is Chapter 2 of the Third Edition of the Complete Digital Photography by Ben Long, though the mistakes here are probably mine.

For further reading:

How Stuff Works answers how a camera works
How an image sensor works?
CCD vs CMOS
Wikipedia's CCD entry