Adventures of a Toronto Student

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[torontostudent]

What can be done about prion diseases?

It’s probably more effective to target the spread of prion diseases rather than trying to stop people from getting mutated prions. Since just one or two misfolded prions doesn’t really matter in your body if they can’t convert the other normal proteins into the diseased form. One possible way to stop these is to engineer some kind of drug that has a 3D shape very similar to PrPsc but can’t turn PrPc into PrPsc. This way it can bind to the growing ends of PrPsc and effectively stop them from latching onto anything else or growing into fibrils.

As a final aside, here are some other distinctive traits of prions and a general summary of what we’ve learned about them. In humans, the prion gene is located in the short arm of chromosome 20. The normal prion, PrPc, has more alpha helical structures (longer alpha helices), while the diseased prion PrPsc has many beta sheets. Since they’re a cellular protein, the cell doesn’t exhibit an immune response towards them. The diseased form of the prion is due to a different protein conformation, and right now even though their mechanisms seems similar to some of the stuff in other diseases, they are not officially identified as the cause of Alzheimer’s, Parkinson’s, and Amyotrophic Lateral Sclerosis (ALS).

We call the diseased form (PrPsc) an isoform of PrPc with high beta sheet content because they are technically still the same protein but just have different post-translational modifications. PrPc has almost no beta sheets in its secondary structure. The diseased form PrPsc is also resistant to digestion by proteases (proteolysis). 25% of the cases of known prion diseases have been caused by inherited CJD (Creutzfeldt-Jakob Disease), GSS, and FFI (Fatal Familial Insomnia). The inherited forms are caused by mutations in the gene that cause the amino acid sequence to change when they make the PrP protein from the cellular genome.

The aforementioned prion disease GSS in the last paragraph stands for Gerstman-Straussler Syndrome. The inherited form causes the normal proline at codon 102 to turn into the amino acid leucine instead. In Creutzfeldt-Jakob Disease, the mutation is at codon 178, where the normal form’s Aspartic Acid (D) changes to Asparagine (N) in the diseased form. In Fatal Family Insomnia, the amino acid D178 (Aspartic Acid in codon 178) changes to Asparagine (N). The prion protein “replicates” by using PrPsc as a “seed” to convert normal PrPc into PrPsc. Specific mutations in the protein can also convert it into a diseased prion. These infections are very slow and may incubate from months to years before any symptoms show.

[torontostudent]

Creutzfeldt-Jakob Disease (CJD)

Creutzfeldt-Jakob Disease will be our case study about prion protein diseases. CJD is the best known human prion disease. It infects one in a million people every year (i.e. 1 person in the world gets infected for every million people there are). This means that in Canada there are around 35 new cases each year. Since it’s a prion disease, CJD can also be divided into the genetic, acquired, and sporadic types. This just describes the method in which the individual got the disease, but the mechanism of action is still the same in each of them.

Sporadic CJD occurs unpredictably and accounts for over 90% of Canadian CJD cases. As for the genetic case of prion disease (caused by having a genetic mutation in the prion gene), at least 7% of prion disease cases in Canada are caused by this. Acquired prion disease (i.e. getting it from eating infected cattle or from other people) are the rarest form of transmission in Canada. The transmission sometimes occurs through accidental transmission during medical procedures, known as iatrogenic CJD (don’t skimp on sterilization routines for medical tools! Prions are hard to destroy!). We have a new term for CJD that is acquired, and we call it variant Creutzfeldt-Jakob Disease (vCJD). The variant form mainly affects younger people and is caused by exposure to food that has been contaminated by Bovine Spongiform Encephalopathies (mad cow disease). Even though vCJD has occurred in Canada, it’s extremely rare.

As for sporadic CJD, many cases of sporadic CJD arise in people with specific forms of the normal PRNP gene. These forms are the 129M and 129V forms, and they occur widely in the human population. This is different from genetic CJD because these variants do not cause the prion disease (in genetic CJD they actually have genes that produce diseased proteins), but people with these normal PRNP genes are more likely for some reason to have a spontaneous mutation in their CJD protein. Each person has two copies of their genes, one from the mother and one from the father. Thus we can describe the PRNP genotype as MM, VV, or MV depending on their combination of 129M and 129V genes. When they looked at the people who have sporadic CJD, they saw that 70-80% of the patients had the MM genotype. We don’t know why the MM genotype is more likely to get sporadic CJD though, but for now we just know that it’s more important for people with the MM genotype in PRNP to be aware that they’re at higher risk of developing Creutzfeldt-Jakob Disease.

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Prions - Mechanism of Action

We talked before about how the Scrapie form of PrP has a GPI anchor in it. This means that it attaches itself onto the cellular membranes, and most of the body of the protein is found in extracellular fluid. Normal PrP proteins are produced in the endoplasmic reticulum (ER) and then shuttled to the golgi, where they enter vesicles and are transported to the cell surface. Once they reach the cell surface, the scrapie form proteins swoop in and latch onto them with the help of cofactors like GAG which bind to both the normal and scrapie forms. The normal form ends up being folded into the scrapie form and they accumulate on the cell, turning into amyloid plaques and fibrils. Sometimes these misfolded proteins get endocytosed and the endosomes turn into lysosomes because the cell is trying to get rid of them, but the lysosome doesn’t work on the misfolded forms so you just get a bunch of lysosomes filled with scrapie PrP proteins.

When a normal proteasome digests / gets rid of a protein, it can only act on proteins that have been ubiquitinated (marked for destruction). It digests the protein and the ubiquitin detaches and goes around to mark more proteins for destruction. For PrPsc though, it binds onto the active site of the 26S proteasome but nothing happens. This is bad, because now with a PrPsc protein bound at the active site, the proteasome can’t go around digesting more proteins, and all the proteins that were supposed to be destroyed ends up accumulating in the cell.

Other proteins also involve aggregation of proteins into amyloid fibers, but we just don’t call them prions. These diseases include type II diabetes, Alzheimer’s, Parkinson’s, and Huntington’s disease. There are also proteins that can self-propagate (trigger their own creation) as well in yeast and fungi. These self-propagating proteins serve biological functions such as modulating RNA processing and actually do not harm the yeast and fungi.

The hypothesis we have now about how prions bind is that first they trigger the formation of PrPsc from normal PrPc, and then the PrPc join together side by side to form a chain of two units, then three units, etc. Then we have larger groups of prion chains joining together to increase the chain even faster. The main idea behind this is that longer chains of prions don’t grow one by one, but instead form smaller sub-chains and then the sub-chains join together to form the long chain. In the variant Creutzfeld Jakob Disease, we suspect that abnormal prion proteins from cattle enter into the human body and convert the normal PrPc forms of the new human host into the diseased form.

Sporadic CJD can occur because sometimes due to translational errors or spontaneous gene mutation, it can just happen that the mutation causes the normal PrP protein to convert into the diseased PrPsc form. Then that one protein converts the others around it and leads to sporadic CJD. This is very rare because translation and transcription errors can occur at any place in the gene so you’d have to land that very small chance of getting just the right mistake to turn the PrP protein into a deadly form.

There’s also genetic prion disease, where the individuals have a mutation in their entire genome at the PRNP gene. If people have a mutation in their gene, their PrPc proteins will be more likely to be converted into the PrPsc form.

[torontostudent]

Genetics, Prion Protein Structure and Function

So, the normal form of the prion protein is also called PrP 33-35 and the disease form is PrP 27-30. After translation, PrP 33-35 has to undergo some post-translational modifications to become PrP 27-30. The numbers just means that before the modifications PrP is 33-35 kiloDaltons in weight and then after the modifications PrP is 27-30 kiloDaltons in molecular weight. PrP has to be glycosylated at two sites (sugars are added to it). Two cysteine residues on the protein have to make a disulfide bond between each other. The N-terminal signal peptide has to be removed (signal peptides on the N terminus are like a shipping label for the protein so the cell knows where it should go). A hydrophobic (tries to get away from water) segment on the C-terminus of the PrP protein also has to be removed. A phosphatidylinositol glycolipid has to be attached to the C-terminus (this is commonly called a GPI anchor). Then the first 57 amino acids at the N terminus are removed. After all of these have been done, then a normal PrP protein is now folded like a diseased PrP protein.

In terms of secondary structure, the main difference between the scrapie form of PrP and the normal form is that the scrapie form contains more beta sheets and fewer alpha helices. The 3D structure of PrP 27-30 (diseased form) contains two alpha helices on top of a long triangular prism of beta sheets. The GPI anchor in the PrP protein lets it anchor into the plasma membrane of cells, and the peptide repeat sequences typically bind onto copper ions (Cu II). The 3D structure of a normal prion protein doesn’t have the long rectangular prism of beta sheets and instead just has longer alpha helices.

Normally, only PrP 33-35 proteins should be made in our cells. They can be found in the neural cell membrane where they’re supposed to bind to Cu II ions as their main function (Cu2+). However, when PrP 27-30 proteins are produced in diseased cells, they end up being able to trigger their own production from normal proteins. This means that they can turn part of the alpha helix in PrP 33-35 into the beta sheet triangular prism permanently. Then the PrP 27-30 proteins with many beta sheets group together to become amyloid fibrils. These amyloid fibrils kill neurons in the thalamus by inducing apoptosis (making it kill itself).

Now, for the functions that the normal prions are supposed to carry out in the cell, they studied this by making PrPC.knockout mice and conducted experiments on them (scientists stopped PrP proteins from being produced and then observed the mice to see what happened to them). The first observation they made was that the circadian rhythms and sleep patterns of the mice became messed up, indicating an inability to regulate sleep. Then they found that there was a lot more oxidative stress in the mices’ brains. There wasn’t enough copper in the brain (PrP proteins bind to and keep copper in the brain), and the activity of super oxide dismutase (SOD) was also reduced. Super oxide dismutase reduces oxidative damage, so decrease in the activity of SOD was also found alongside increased oxidative damage in the brain. There was also a reduction in the amount of mitochondria (you all know the meme “mitochondria is the powerhouse of the cell”, i.e. less mitochondria = less energy to work with in the cells).

Other observations in these knockout mice were found in the immune system. Phagocytosis was increased (this is usually to swallow up enemies). Interestingly, mice that did not express PrP proteins had increased resistance to Brucella abortus (bacteria) and the herpes simplex virus-1 (HSV-1). There were also increased levels of interleukins expressed in the cell (interleukins regulate the immune response). We still don’t know what exact roles PrP plays in the immune system that would induce these changes though. In neurons without PrP, they had problems carrying out long term potentiation (i.e. the neurons without PrP can’t do some of the things that they normally should be able to do). The mice had increased susceptibility to seizures, and the mossy fibers in the hippocampus became disorganized.

As for behavior of the mice, they exhibited cognitive defects and impaired memory, along with increased exploratory / locomotor activity (they moved around a lot more). We think that the reason why the mice moved around a lot more is due to MK 801, which is supposed to help stop convulsions. The mice also had less symptoms of anxiety but were more susceptible to Dpl (Dopple toxicity - dopple is a protein similar to PrPc). They were also susceptible to toxic effects caused by “delta F PrP” (a shorter form of PrP). Other things that they had increased susceptibility to were ischemia (restricted blood supply), apoptosis death of cells caused by ethanol, and traumatic brain injury.

Some other effects of getting rid of PrP was the impaired self-renewal of hematopoietic stem cells (i.e. they get less blood stem cells, this may help to explain why they were more susceptible to ischemia). There was also a decreased generation of neural precursors (less baby neurons). There was also less dentin in their teeth (dentin is a calcified tissue, and one of the four major components of teeth).

All in all, the takeaway should be that PrP protein is involved in a lot of bodily functions and even though it can be bad for us if they get misfolded, we can’t just get rid of all PrP entirely.

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Prion Pathology (Symptoms, etc.)

The main differences in terms of pathology between the normal prion protein form PrPC and the diseased form PrPSc is that the Scrapie form is resistant to cellular processes that were supposed to break down this protein and remove it from the body. This resulted in having no way to get rid of / recycle this protein and it ended up being accumulated in the brain of patients with a prion disease.

There have been many other terms for these accumulations of prion proteins in the brain. Sometimes they’ve been called amyloid plaques. These amyloid plaques are clumps / aggregates of proteins that are very resistant to breakdown and removal. Other neurodegenerative disease have also identified plaques consisting of misfolded proteins, such as in Alzheimer’s disease, and also in normal aging brains. However these diseases have plaques made from proteins other than the PrP protein. This just means that the PrP protein is probably the first well-studied prion disease but many other proteins in the brain may behave similarly.

When you look at prion diseased tissues through a microscope, there are two distinct forms you could see. The spongiform encephalopathies are clusters of vacuoles that look like a bunch of empty bubbles smushed together. There are also clusters of prions, such as in kuru plaques (kuru is another prion disease). These appear like a bunch of dark dots gathered in one area.

Since prions affect the brain, prion diseases are essentially diseases of the central nervous system. In kuru disease, the symptoms include trembling, difficulty walking, trouble with bodily coordination, dementia, etc. All of these result from faults in the brain which is your area of motor control. The symptoms in Creutzfeld-Jakob Disease (a rare type of human dementia) and Gerstmann-Straussler disease are also attributed to prions.

In sheep infected with Scrapie disease, the sheep scrape their wool against the fences. This disease is also caused by prions like we’ve mentioned before. However, as of now, there is not enough research yet to firmly conclude that diseases such as Alzheimer’s with symptoms of dementia, lack of coordination, difficulty walking and other movements resulting in death, is necessarily caused by prions. They are probably involved though. The same goes for Parkinson’s disease which has symptoms like ganglion cell destruction, neurofibrillary degeneration, and dementia. Another neurodegenerative disease with similar symptoms is Amyotrophic Lateral Sclerosis (ALS), which results from the destruction of motor neurons.

Since these diseases all have similar pathology and clinical signs, it is possible that they’re all related, or even variants of the same disorder. All the pathological features (disease manifestations) are in the central nervous system, and we know that prion proteins are selective for the central nervous system and accumulate in CNS nerve cells. For PrPSc, it accumulates in the neurophil. This causes astrocyte gliosis (increase in # of astrocytes, which is a star-shaped glial cell), depletion of dendritic spines in neurons (neurons are less interconnected, probably can’t receive signals from other neurons as effectively), and the formation of vacuoles in the cerebellar cortex. These vacuoles are essentially empty holes in the brain that we call spongiform encephalopathies. The scrapie prion protein also causes amyloidosis, which is the increase of amyloid in the cerebellar cortex, thalamus, brain stem, and lumen of blood vessels found in the brain. Amyloids are aggregates (groups) of proteins that stick together. These amyloid plaques are eosinophilic, meaning that they can be stained by eosin in experiments and then subsequently visualized. The plaques often have amyloid fibrils radiating out at the sides. The amyloid fibrils are still just collections of proteins but they take on a ore stringy structure instead of being a large chunk. The plaques are subependymal, subpial, and perivascular (the first two mean that the position of these protein plaques in the brain are under the ependyma which is a thing in the brain, and also under the pia mater which is also another part of the brain. Perivascular means that it’s usually found around the blood vessels).

One huge difference between prion disease pathology and virus pathology is that prion diseases do not result in any inflammation of fever! This is very important, because fever and inflammation only occur as a result of the immune system going into action. This means that the immune system is not responding to these prion proteins. This makes sense when you think about it, because diseased prion proteins are derived from normal proteins that result from everybody’s genome.

The symptoms of prion disease also result from the disease pathology described above. There is a long incubation period of several years, which has been coined a “slow infection”. There is also loss of or abnormal function in the cerebellum, resulting in difficulty walking and loss of muscle coordination. Dementia also results later on, which involves loss of memory, diminished intellect, and poor judgment. Patients may also see progressive insomnia with either reduction or loss of the slow-wave and rapid-eye-movement phases in sleep.

Here are some quick examples of prion diseases. There’s scrapie which infects sheep and goats, and it’s transmitted from sheep to other genetically susceptible sheep. Transmissible Mink Encephalopathy (TME) infects Mink, and they get it from sheep or cattle with a prion disease. Chronic wasting disease is present in mule, deer, and elk, and right now we don’t know how they get it. Bovine Spongiform Encephalopathy (BSE) infects cattle, and they get it through eating prion-contaminated feed (usually bonemeal, i.e. ground bones from infected sheep which are put into their feed in order to give them more protein to grow etc.). In humans, there is kuru disease which is a prion disease that transmits in certain tribes due to them having rituals where they cannibalize deceased relatives and tribe members in order to let them move on in peace. Other human diseases are Creutzfeld-Jakob disease (CJD), Gerstmann-Straussler Syndrome (GSS), and Fatal Familial Insomnia (FFI), which result from a germline mutation in the PrP gene (i.e. passed on genetically). Then there’s the variant form of Creutzfeld-Jakob Disease (vCJD) in humans which is believed to be transmitted through eating infected beef.

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History of Prion Research

As for prion research, they started by trying to study scrapie infectivity in mice through the use of sedimentation properties. They didn’t really learn anything for this except that infectivity was spread throughout the entire gradient - i.e. their method of measuring for infective particles didn’t work on scrapie (didn’t separate the infective particles cleanly). Thus, different research methods had to be devised.

In in vitro studies, they tried to inject brains of scrapie-infected sheep into lab mice, but found that it took another 1-2 years of inoculation for the mice to succumb to the disease and become available for data collection. This was too slow for the scientists, but then they discovered that syrian hamsters succumbed much more quickly to the scrapie disease. Thus they began experimenting on syrian hamsters instead for scrapie studies.

They found the phenols, which could hydrolyze or modify proteins, reduced scrapie, but the disease was resistant to treatment methods that altered nucleic acids (UV radiation). Then finally the hypothesis that scrapie was a protein emerged. Thus they coined the term prion, based on how it was proteinaceous and infectious.

PrPSc (Scrapie form of Prion Protein) was identified from the brains of sheep infected by scrapie. They tested portions of sheep brain to see if they were capable of being infected, and they were. The cellular form has superscript C and is not infectious. This prion protein has a molecular mass of 27-30 kiloDaltons and is the protease-resistant core of PrPSc. They managed to sequence the N-terminus of the protein through Edman degradation (way of sequencing where you cleave off amino acids one by one in order to get the correct order). This allowed them to reverse predict the nucleotide sequence and then conduct molecular cloning experiments.

Interestingly, they found that mRNA levels for this gene were the same in infected and uninfected animals, which meant that the disease wasn’t being spread on an mRNA level. They also found a protein that was from the same sequence in animals that was sensitive to protease (the disease protein was not sensitive to protease). They named this isoform (same sequence, different 3D structure / alignment) the PrPC protein. Afterwards, cDNA probes became available (complementary DNA) and using these cDNA probes they were able to find where in the body these PrP proteins were being expressed. The result was that the PrP proteins were expressed in the brains of uninfected adult mammals.

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Prions - Introduction

Prions are infectious pathogens that cause fatal neurodegenerative diseases by an unprecedented mechanism. In prion proteins, the prion protein (PrP) is modified. Prion diseases can appear either genetic, infectious, or sporadic. Some notable prion diseases are Bovine Spongiform Encelophalopathy (BSE), Scrapie in sheep, and Creutzfeldt-Jakob Disease (CJD) in humans.

Prion diseases were big in the news a few years ago when the Mad Cow Disease garnered lots of media attention. Officially called Bovine Spongiform Encephalopathy (BSE), mad cow disease was a prion disease that swept through the cattle industry in the UK. The breakout led to fear of transmission to humans through dietary exposure in beef and beef products.

Although there is still no proven causal link between Bovine Spongiform Encephalopathy(BSE) and a novel form of Creutzfeldt-Jakob Disease (CJD), this mad cow disease prion could be a likely explanation. This is because in recent years a new form of the Creutzfeldt-Jakob Disease has arisen, called sporadic Creutzfeldt-Jakob Disease.

Fundamentally, prions are just proteins. These proteins can cause fatal brain diseases. There are human prions as well, and those can be transmitted or inherited. These infectious prion disease particles contains protein but no nucleic acids (i.e. no DNAs, no RNAs). Human prions are also a part of normal brain function so they have a host gene that encodes the normal form of these proteins. The prion hypothesis is that the prion disease particles are alternative forms of our normal protein, and these alternative forms are actually not folded correctly (proteins fold to form a 3D structure). This misfolded prion protein can make normal proteins refold into the ‘bad’ form. Right now, it’s theorized that prion proteins probably have something to do with Alzheimer’s and Parkinson’s.

We call prion diseases transmissible spongiform encephalopathies. These are neurodegenerative conditions that affect humans and animals, meaning that you won’t see any symptoms immediately but they’ll slowly erode your brain matter until you are much older and decreased brain functions are observed. Usually, transmission is through inoculation (the prion goes inside of you) or more rarely, dietary exposure to infected tissues (through eating it).

We thought that transmission of prion diseases between species was unlikely due to the species barrier, which just means that since animal species are so different it would be unlikely for diseases to be able to infect multiple animal species with one strain. However, this species barrier was broken when Scrapie transmitted from sheep to mink (carnivorous mammal, looks like a ferret). This happened because mink ate some processed meat from sheep with Scrapie disease. This resulted in the Transmissible Mink Encephalopathy (TME) which basically stopped all mink farming efforts. The recent resurgence of bovine encephalopathy in the cattle industry (mad cow disease) seems to also have arisen through feeding cattle with processed meat that originated from sheep infected with Scrapie.

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siRNA Technologies in Plant Science

In plant science, siRNAs are inserted into the plant through either viral vector delivery (the virus should be a retrovirus so the gene integrates into the plant genome) or by just making a transgenic plant that expresses the designed siRNA. Effects that siRNA expression can induce in plants by knocking out genes are: resistance to plant pathogens such as insects, viruses, and nematodes. It can also allow the genetic engineers to control fruit ripening and flower development. You can also use an siRNA spray as insecticide. Knocking out genes can also affect plant fertility, sterility, and longevity.

An example of virus induced gene silencing in plants is the use of Tobacco Rattle Virus in the N. benthamiana plant. First you have to select the gene sequence for cloning and then inserted into pTRV2. pTRV1 and pTRV2 are just viral vectors based on the Tobacco Rattle Virus. pTRV2 has to contain the gene to be inserted while pTRV1 has the RNA-dependent RNA polymerase, movement protein, etc. Both of the plasmids have to be inserted into Agrobacterium tumefaciens, a type of bacteria that you use to deliver the plasmids into the plant. You can use a toothpick to prick the plant open on some sites and then inject the A. tumefaciens into the plant with a syringe. Then the plant is put into a vacuum (insulated space) and after waiting for 2 to 3 weeks the desired phenotype should be observed.

Another example of siRNA technology to make transgenic plants is the use of siRNA technology to make plants resistant to grapevine fanleaf virus. In these experiments, they made transgenic rootstalks, which is basically the bottom half of the grapevine plant which is under the soil. The top half is called the scion and that’s where the leaves are. These siRNA have a hairpin shape and they target Grapevine FanLeaf Virus (GFLV). It’s possible to only genetically modify the rootstalks with the siRNA and then the silencing RNAs end up passing from the rootstalks into the non genetically modified scions. This demonstrates that siRNAs can pass through grafts. This lets farmers end up with non transgenic grapes (not genetically modified) and yet also Grapevine FanLeaf Virus (GFLV) resistant.

When designing the transgenic rootstalk, the hairpin siRNA has to loop around on itself so it needs to contain a reverse complementary sequence. The part matching the sense strand has to be engineered to be on the 5’ end of the siRNA, and the antisense strand sequence has to be on the 3’ end of the siRNA. As a side note, wine contains no DNA from the plant, only juice from the plant.

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siRNA Applied Technologies

Another way to deliver siRNA into cells is through the use of nanoliposome delivery vehicles. Nanoliposome delivery vehicles mimics the cellular phospholipid membrane. It’s basically a bubble of fat containing the siRNA inside it. You can also put drugs or DNA inside it as well. This is made specifically to target mammalian cells because plant cells have a cell wall instead of a cell membrane which defeats the whole purpose of using a lipid membrane to make fusion into the cell easier.

These nanoliposomes contain a protective layer on the outside to stop it from immune destruction (probably ligands or other things inserted at the surface of the phospholipid membrane). There are also homing peptides inserted into the liposome’s lipid bilayer membrane with their ends sticking out from the surface. These homing peptides are surface ligands which target and make sure that the liposome only binds and enters into diseased or targeted cells. We can design these surface ligands to be specific to certain types of receptors that are only found on the cells we want to target.

The aforementioned two techniques to deliver siRNA into the body have already been applied to some diseases. Scientists have found gene products that are associated with Hepatitis B and C, cancers, autoimmune disorders, neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. Silencing RNAs have been developed against these pathogenic gene products and introduced into mammalian cells using viral vectors (lentivirus) or nanoliposomes.

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Applications for siRNA in Biotechnology

Since siRNAs have this RNA suppression capability, scientists have started to use them to knockout genes at the RNA level in experiments. The purpose of these knockout experiments are to identify the functions of a particular gene in a biological pathway. Basically, they see what changes when a gene stops being expressed in order to find out what it does. This can give us more knowledge about biological pathways that haven’t been extensively investigated, or help identify genes that play a role in certain diseases like cancer. But there remains a problem. How can we deliver siRNAs to tissues in these experiments?

Ultimately, what we want to do is insert RNA into the cytoplasm of cells in a specific target organ. We can’t use systemic delivery (usually involving getting the particle into the bloodstream) because naked RNA can’t go through the cell membranes. Additionally, there are RNases in the bloodstream which will digest these naked RNAs. Even if we chemically modify the siRNAs to be more stable, it’ll still only have a half-life of at most a few hours. This is why we need other ways to deliver these siRNAs into the cell.

However, there are several challenges to finding a good way of delivering siRNA into the cell. For example, the delivery vehicle we use to deliver the siRNA could end up being toxic to the cell so we have to make sure that isn’t the case. We also need to make sure that the siRNA doesn’t end up being degraded by other enzymes in the serum before it makes its way to the target cell. Finally, we need to make sure that the siRNA can still work as intended and that it won’t suppress any non-target genes (i.e. act nonspecifically). Suppressing non-target genes is bad because then it defeats the whole purpose of using siRNAs to study the effects of specific genes (since the observed effects could be caused by other genes that the siRNA ended up suppressing).

Ultimately, the method of delivery that we settled on was to use the lentivirus. Lentivirus is a retrovirus, so it uses reverse transcriptase to integrate its linear single stranded positive-sense RNA into the DNA genome of the infected cell. These viral vectors have been modified so that they are replication-deficient, in order to minimize the risk of injecting them into the body as a treatment method. This is because these lentivirus vectors carrying siRNAs that have been modified into their genomes are meant to be used on mammalian cells, meaning that they could potentially become a treatment method in the future so it’s important to ensure that they are safe.

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Virus Suppressors of Gene Silencing

In plants, RNA viruses trigger RNA induced silencing, which is normally very effective against the viruses. However, viruses developed a way to combat the RNA induced silencing. For example, the potato virus X (PVX) contains a 25 kiloDalton protein involved in viral transport. This viral transport protein strongly represses gene silencing. Another virus that has a repressor for gene silencing is Potato Virus Y (PVY). Potato Virus Y has a protein called the Helper Complement protein which acts as the gene silencing repressor. Many other plant viral proteins also have the ability to repress RNA silencing.

Another acronym that they invented for the RNA induced gene silencing when it’s specifically being used to combat viruses is VIGS. VIGS stands for Virus Induced Gene Silencing. These suppressor proteins in viruses have many different mechanisms which target different steps of the antivirus silencing pathway, which suggests that these viral counter-defensive strategies probably evolved separately from each other. The proteins which have the anti-VIGS component are also different in each virus. For example, CMV has it on a movement protein while CPMV has it on the small coat protein, etc.

Let’s see the variety of different steps in the RNA interference pathway at which viral proteins can act. The 2b movement protein of the Cucumber Mosaic Virus (CMV) blocks the amplification step where RNA Dependent RNA Polymerase in the cell makes more dsRNA based off of the CMV RNAs that have been sliced up by RISC. This 2b movement protein additionally also acts on the RISC assembly step, which occurs after the viral RNA is sliced up by dicer. Thus, 2b also prevents the Argonaute bound to RISC from taking in a template RNA strand.

Meanwhile, the Helper Complement Protein (HC-Pro) of Potato Virus Y (PVY) acts only on the RISC assembly step in order to stop the RNA silencing pathway. HC-Pro does this by blocking RISC activity rather than affecting the actual assembly of RISC (some other proteins stop the actual assembly of RISC by having proteins that protect the viral siRNA from being taken in by the argonaute on RISC). So in PVY, the siRNAs can enter the RISC complex but HC-Pro binds onto one of the active sites of RISC to stop it from latching onto viral RNA.

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RdRps and Gene Silencing

RdRp stands for RNA Dependent RNA Polymerase. In experiments with RNA silencing, they observed that RNA silencing happened by even just injecting a few molecules of double stranded RNA per cell. This RNA silencing was able to spread throughout the entire animal or plant, and persisted from the parents that were injected with double stranded RNA to children that weren’t treated with double stranded RNA. This suggested that the few strands of double stranded RNA might have been copied into more double stranded RNA, which is how the RNA silencing spread so quickly (more templates to silence off of). Since only RNA was injected, there must have been RNA Dependent RNA Polymerase (RdRp) in the cell which made more copies of the RNA. There were also experiments with transgenes that were being silenced (transgenes are artificially inserted genes). They thought that silencing for these transgenes could occur through RdRp transcribing a complementary sense RNA which was used as a template to silence these transgenes.

RNA induced silencing has two functions at the DNA level: to inhibit transposons from jumping around and wreaking havoc in the genome, and to silence the transcription of chromosomal genes. In some viruses, double stranded RNA needs to be generated as a replicative form, and that triggers the dicer enzyme to degrade the dsRNA into fragments which creates more siRNA. As mentioned above, the cellular RNA Dependent RNA Polymerase uses the siRNA as primers to generate more copies of foreign double stranded RNA, which then spread throughout the cell and go through the dicer and RISC pathways again to bind to corresponding mRNA. The RdRp can also extend sequences to generate more double stranded RNA once it locates the whole strand that siRNA is bound to. This results in even more effective degradation of viral RNA in the cell as a result of gene silencing, and viral infection should thus be reduced. This is a special antiviral process in plants.

Summary: Small RNAs

These RNA-mediated silencing pathways have been conserved throughout evolution since they are found in such a wide variety of plants. Studying RNA interference pathways helps us to understand eukaryotic gene regulation and learn more about how eukaryotic hosts defend against viruses and transposons.

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3 Basic RNA Silencing Pathways in Higher Plants

The three RNA silencing pathways in higher plants (generally those are not algae, i.e. more ‘advanced’ plants) are condensation of chromatin into heterochromatin, virus induced gene silencing (defense mechanism), and the use of miRNAs for the regulation of plant gene expression.

The condensation of chromatin into heterochromatin is just winding the DNA more tightly on the histone ‘spools’ so that nothing can come and transcribe it. Generally, DNA that is loose and easy to transcribe is called euchromatin. Euchromatin has a lot of acetylation on this histones. Tightly packed DNA is called heterochromatin, and it is highly methylated. Some small RNAs can actually go into the nucleus and promote the formation of heterochromatic DNA, blocking transcription. For example, the methylation of cytosine in DNA results in reduced gene expression that is maintained through cell division (because the DNA is the same).

An interesting note to add to the siRNA silencing pathway (exogenous RNA interference) is that the siRNAs can actually act as a mobile silencing signal. If siRNAs float between the plant cells from one cell to the neighboring cell, the RISC proteins from the neighboring cell can pick up the siRNA and then use it as a template to silence viral RNA there as well.

For the RNA interference mediated chromatin modifications, the siRNA that goes back into the nucleus is not bound to RISC but is instead bound to RITS (although the siRNA is still formed through cleavage by dicer protein). RITS stands for RNA Induced Transcription Silencing, and it’s also a protein complex but I’m guessing they probably dropped the C since it doesn’t make for a good acronym. The single siRNA strand and RITS bind together and go into the nucleus, where RNA is being transcribed on DNA. RITS tries to find RNA that is currently being made which has a complementary sequence to the siRNA, and that’s also where the target DNA gene should be. It cleaves the RNA that is being transcribed and recruits a bunch of methylation factors over to methylate both the histones and the DNA, which turns it from euchromatin into heterochromatin.

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Functions of RNA Interference in the Cell

The two main functions of RNA interference are to defend against foreign genetic material, and to regulate cellular gene expression. For defence, the foreign genetic material usually comes from viruses and other pathogens that are capable of making their own double stranded RNA molecules in the cell. These pathogens can usually take advantage of proteins and processes in the infected cell to aid their own replication, so RNA interference may have been originally used to slow down any processes that have been hijacked by foreign RNA.

As for the regulation of gene expression, the roles of RNA interference differ between plants and mammals. In plants, miRNAs usually regulate transcription factors. Thus, miRNAs are found in many cells in plants, and they control the expression of regulatory genes that are involved in plant development (i.e. they do a lot of stuff because basically every gene is controlled by transcription factors).

In humans and other mammals, micro RNAs have also been found in tumor cells where the cell cycle has gone haywire. Thus, they could potentially play roles as both oncogenes (causing cancer) and tumor suppressors (preventing cancer). There are many miRNAs in the cell so that’s why we can’t really say that they are definitively good or bad, since some miRNAs may end up helping the cancer spread while other miRNAs could be working towards stopping it. It’s like how we can’t really say that humans are good or evil since there’s such a wide variety of them. However, we just do know for certain that they are found in cancer tumor cells so have something to do (either good or bad) with cancer.

We call the RNA interference pathway where they suppress foreign genetic material the Exogenous Pathway, and gene regulation is the Endogenous Pathway. The only major difference in these pathways occurs before the RNA Induced Silencing Complex (RISC) gets involved. The endogenous pathway involves the aforementioned pre-miRNAs that were transcribed in the nucleus and then exported to the cytoplasm, while the exogenous pathway involves foreign double stranded RNA that is found in the cytoplasm. The dicer enzyme and RISC are both found in the cytoplasm, and once RISC gets the small section of template RNA that it needs, it can proceed to either exogenous or endogenous gene silencing. In exogenous gene silencing, the small RNA template used by RISC is generally called small interfering RNA, while in endogenous gene silencing we have a single stranded RNA that forms a stem loop hairpin structure, and when it is taken up by RISC it’s called a micro RNA.

Thus, the main differences between siRNA and miRNA is that the siRNA is from outside the cell while miRNAs are from inside the cell. SiRNAs are double stranded and usually from a viral genome or an inserted gene in genetic modification (those also commonly use viral vectors for delivery so it counts as a ‘viral gene’). MiRNAs are actually single stranded RNAs that base pair with themselves after they loop around. These are usually the introns that get spliced out and then taken up by the dicer protein. Both siRNAs and miRNAs are used as templates to match targets in the RNA Interference pathways.

siRNA Pathway

Once the double stranded viral RNA is cleaved by the dicer protein into 21 base pair chunks and then one of the chunks is taken up by RISC, one of the two strands of RNA is degraded. This is because RISC only uses one RNA strand as the template. This way, the RNA strand template is actually capable of base pairing with complementary sequences. The siRNA latches onto any mRNAs with a complementary sequence section, which are hopefully all viral mRNAs (positive sense). This prevents the mRNA from being used as a template during translation. So, as soon as it binds on, we can see that the viral proteins are not being made anymore, thus the RNA is ‘silenced’.

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Major Players of RNA Silencing Pathway

The major components involved in RNA silencing are the Dicer protein, RISC (RNA Induced Silencing Complex), and the argonaute protein (AGO). Dicer is involved in the creation of miRNAs from the primary miRNA transcript. Micro RNAs also have to be created from their initial RNA transcript. The primary miRNA transcript has branched hairpin structures on it, and the miRNAs come from the stem of these hairpin structures. The stem contains RNAs hydrogen bonded to each other (the same RNA loops around and then base pairs with itself). This mimics a double stranded RNA structure, which makes the dicer protein come and digest it. However, the dicer protein doesn’t digest double stranded RNAs into single amino acid subunits. It just cuts them up into smaller chunks of 20 to 25 base pair double stranded RNAs. (The dicer protein is an endonuclease which is why it can start to digest from the loop of the hairpin structure. Apart from being active against pre-miRNAs, dicer is also active against viral double stranded RNAs, since usually the cell isn’t supposed to have any double stranded RNAs which is how it just kind of assumes that any dsRNAs are foreign viral genomes and it has to cut them up to defend the cell. The dicer protein also leaves an overhang of two nucleotides on the 3’ ends of the double stranded RNA fragments that it spits out (due to the cleaving mechanism).

After the smaller chunk of double stranded RNA is spit out by the dicer protein, one of the two strands is selected by the argonaute protein (AGO). Argonaute is part of the RNA Induced Silencing Complex (multiprotein complex), and possesses RNase activity (can digest RNA). This one strand from the double stranded RNA becomes the miRNA. Thus, in a way, the selection by argonaute is what finalizes which side of the hairpin loop becomes the miRNA. Since the main target for silencing is supposed to be RNAs that will be translated into proteins, argonate will select antisense (negative sense) RNA strands instead of the sense strand, since only sense strands can be translated and we need a template that is complementary to them.

The RNA Induced SIlencing Complex (RISC) is 200 to 500 kiloDaltons of proteins. It’s a multiprotein complex that takes a strand of siRNA or miRNA to use as a template for recognizing complementary mRNA or viral RNA. That’s why it goes and latches onto digested proteins from the dicer, because it knows that anything that the dicer has digested should be a good target (probably foreign) to destroy in order to help the cell. Thus, it takes one of the strands to find the any complementary sequences in the cell, and once it’s able to base pair onto a complementary RNA strand the RNase activity of RISC is activated and it cleaves the RNA, destroying it. Even when it hasn’t destroyed the mRNA yet, latching onto the mRNA is still able to block translation of the base paired segment.

Dicer Protein

The dicer protein is ATP-Dependent, so it needs the basic cellular unit of energy in order to carry out its cleaving functions. It functions similar to ribonuclease III, processing both endogenously made and exogenously inserted double stranded RNA into chunks of 21 to 25 double stranded RNAs with a 2 to 3 nucleotide 3’ overhang on each side. This means that the 3’ end is usually a bit longer than the 5’ end (more like a diagonal cut than a straight cut).

In mammals, there’s only a single dicer protein. This is the same in Caenorhabditis elegans (C. elegans, the worm that’s used really often in genetic experiments) and fission yeast (also a model organism in biology). However, there are two dicer proteins in drosophila melanogaster (fruit fly, also one of the most commonly used organisms for experiments). In Arabidopsis thaliana (thale cress, a plant), there are four dicer-like proteins (DCL).

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History of Gene Silencing

In plants, the first to discover gene silencing was David Baulcombe, who discovered that plants exhibit gene silencing as a form of antiviral defence.

Then, Fire and Mello won a nobel prize for investigating gene silencing in the worm C. elegans. Basically, they discovered that artificially injecting gene sequences into cells based on how siRNAs do gene silencing can let the injected gene sequence carry out similar gene silencing effects. This opened up a new research methodology for scientists, since now they could target and silence specific genes and then see what changes in the cell in response to not having the proteins from those genes anymore. The C. elegans worm is also one of the most commonly used organisms in genetic research.

In mammals, Thomas Tuschl’s research group investigated how longer double stranded RNAs can activate an interferon-mediated defence pathway. This is important because now we know that base-paired viral RNA can activate defense mechanisms in mammalian cells.

Discovery of Plant Gene Silencing

We first observed that some transgenic (genetically modified) plants that were infected with an RNA virus seemed to be able to develop some kind of immunity over time. They concluded that the reason for this was some reason other than the immune system recognizing the viral coat proteins. They examined the plant cells more closely and found that both the RNA of the virus and the transgene RNA (RNA of the gene that was planted into the cell by the researchers) were degraded. In other transgenic plants, the gene that researchers planted into these plants were highly expressed as proteins at first, but then the amount of this protein slowly went down. They found that loss of expression was at the RNA level, so something must have happened in the cell for it to realize that there was foreign RNA so it silenced the RNA.

siRNA and miRNA Gene Silencing Pathways

Short interfering RNAs (siRNA) are small RNAs that help both with cellular defense and gene regulation. They act within an interference pathway (a chain of reactions involving a large variety of cellular components like proteins, genes, RNAs, etc.) that involves degrading RNAs that have complementary parts specific to these siRNAs. This means that this pathway is specific and targeted towards the RNAs that are complementary.

Apart from siRNAs, there are also miRNAs. This stands for micro RNA, and they are also short RNA sequences which regulate cellular messenger RNAs.

Origins of Gene Silencing

The reason why siRNAs and miRNAs silence at a post-transcriptional level is because it’s easier to use their sequence complementarity by targeting other RNAs instead of targeting DNAs. SiRNAs work with the help of complexes that have an endonuclease. This is the RISC complex, which stands for RNA Induced Silencing Complex. This short interfering RNA + RISC complex degrades mRNAs with a corresponding sequence, and also viral RNAs in plants. This is called RNAi in animals, which stands for RNA interference. In plants it’s Post Transcriptional Gene Silencing (PTGS) instead. Additionally, in the fungus Neurospara crassa (filamentous fungus) it’s instead called ‘quelling’.

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Gene Silencing

Protein Processing and Degradation

After the protein is translated from the RNA, it could possibly undergo protein processing or degradation in addition to performing its intended functions. In order to degrade a protein, it has to first be ubiquitinated (i.e. have to add ubiquitin to the protein). The proteasome (enzyme that digests proteins) only recognize ubiquitinated proteins, so only ubiquitinated proteins can enter into the proteasome. There, the proteasome digests the protein into peptides (protein fragments), and then the ubiquitin is freed (can be used again) and the proteasome goes off to find another ubiquitinated protein to digest.

RNA Silencing (PTGS)

PTSG stands for PostTranscriptional Gene Silencing, which is just another term for RNA silencing (because after transcription you get RNAs). Gene silencing happens in animals, plants, and some fungi. The actual gene silencing mechanisms are very sequence specific and targeted to RNAs, so that it won’t just end up silencing everything. There are some small non-coding RNAs (around 22 nucleotides in length) called microRNAs (miRNAs) or small interfering RNAs (siRNAs) that are found in plants and many animals. In plants, these small interfering RNAs (siRNA) are used to silence RNAs. The purpose of this siRNA-based RNA silencing is to defend the plant genome against foreign nucleic acids. These foreign nucleic acids could be from viruses, transposons, transgenes, etc. Basically, these siRNAs just want to make sure that only the real plant genome is being translated.