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Unformatted text preview: They7 got the gene for the color and made it a trans gene and transfected the petunia but got these variegated patches petunia. Baulcombe is highly under appreciated. A lot of times people in the animal field don't read the plant literature. when they discoverd this, they looked at these transgenes and they would repress the genes. When they turn on this plasmid, it would actually turn it off. He discovered little RNAs were produced but didn't konwo what they were but they were involved in this silencing. This year, these two guys got the nobel prize for finding this in C. elegans. This is the original experiments that started the field. There was an idea that you can take antisense RNA to a gene and depress the expression of that gene. Sometimes worked and sometimes not. Now, the people that published that throwing in the sense that worked just as well. These guys said maybe the sense and antisense that were coming togethar and the dsRNA was doing this. They got dsRNA to go into C. elegans via injection, eat it, or soak them in it. They looked at several genes and the effect of sense, antisense, or sense nnd antisense and looked at the phenotype. When they put in both, they got the twitches, the mutant. All these genes turned out to be that dsRNA couls someone how repress the expression of a gene as if you were mutating it. Then, this isn't a good experiment, you can't study something until you can do itin vitro. They found Dros. could be effected by dsRNA and you get inject it and see downregulation of genes. They took cycE and lacZ and looked at .....They took Dros embyros and they were transfected with a virus that expressed dsRNA. They goround them up and made an extract. The strip is where they used the RNA. When they used E, they got degradation in a few minutes. If they looked at LacZ there was no effect. It looked like the effect of the RNA was to degrade the mRNA that the RNA was homologous to. The region in white is not effected. It has to be within the region that you use the dsRNA. They decreased the size. There's a minimum size. 100 there was no effect but at 220 there was. They took this lysate in RN interference and ran it on a gel fraction and tested it for degradation of a transcript and what they found was that at this point, 25 nt were made. They got the production of small RNAs ljust oike in Baulcome. 1st, genes could be downregulated by taking dsRNA from that gene and gettign it into the animal somehow. It seemed to work for every organims. It works in mammals but there's problems. Mammals don't like dsRNA and they produce interferon. You don't see any effect, you have to do a trick. There's a high MW complex that is repsoneible for it. What you get are small RNAs 25nt. They looked at them very carefully and weren't 25nt, they had little 2nt overhangs. As soon as they knew there was degradation, they made a model. You start with an exogenous dsRNA, you get cleavage. The complex that they saw was the RISC complex. There's an enzyme that recognizes dsRNA These two strands separate or degraded. The strand that is left is the one that can hybridize. These little siRNA is what does the cleavage. They hybridize and cleaves the RNA and so you get degradation of message. you actually get amplification of the siRNAs because it cuts at the ends. Here's a generalized structure. It cuts int he middle! They also call the antisense strand the guide strand. It's a guide RNA. This grey strand is the one that does the cleaviging. They asked the question, what enzyme recognizes dsRNA and cuts it. We know there's a RNase 3 motif enzyme. They found 3. Dicer, drosha, homeless. DICER had two RIIa motifs,. It had a hlicase motif. Drosha only had the dsRNA binding. Homeless only had the helicase. So they made antibodies and did immunoprecipitation and thn they took what was pulled down and they tested it against dsRNA and they looked for the 25nt RNAs. Drosha had no effect, dicer was litte homeless had no, b gal was control. Dicer is concluded that it is the dsRNA cleavage enzyme. Then they said, does it require ATP? Yes. It requires ATP becdause of the helicase motif. Hydrolysis. The product from this in vitro cleavage, 23nt product co migrates with the RISC product. DIcer seems to be the inital enzyme. It led to this model, it's slightly more domplex. You have dsRNA coming in, you have dicer atp dependent producting two small siRNAs and then there's the helicase that unwinds one end, the end that breathes more, and RISC binds to the strand that is more aactive. It produces an asymetric RNA that can hybridize to the RNA. This ssguide RNA gets assembled into a complex called RISC and an important protein was Argonaut. There were other things but that was very uniform. KO Argonaut lost RNAi. the Guide RNA guides the RISC to the RNA that the dsRNA came from. It causes cleavage and you degrade the mRNA. You can make it in the laboratory. Some of it can be made in the cells. If you have transposable elements, you can get them in the cell. You have dsRNA floating around in the cell. If you're a scientist to study this, you can avoid dicer and throw in synthetic siRNA. Usually you use dsRNA because it's more reesistant to cleavage. I'll come back to the pre miRNA. Then Argonaute. They did a 3D structure and compared it to other proteins. It has a domain called PIWI and it fold exactly like an RNase HI or RNase HII. Maybe PIWI domian has RNase activity. Maybe that's the slicer RNA This is not dependent. It led to this model. Then they tried to prove it. THey tried to purify argonaut protein and in mice there's 2. Turns out 2 is the important one. They immunoaffinity purifie from transiently transfected cells with virus that make dsRNA. This is an artifact but the 3' and 5' products are here. You get cleavage because we just put in 1. It has to be ss. If you put in ssDNA, you get no degradation. It reuires 5'P. No ATP, no effect, so this is not Dicer. This is called slicer. we could do RNAi of argonaut to check it's function. this could be therapuetic. in tryp they put it on a promoter to make tet inducible regulation. You can downregulate any gene in the genome. We haven't talked about why the cells do this. I's great utilit for the researcher AND physician. You can turn off any gene you want including viral genes or cancer genes. There were 2 genes, Lin4, they make RNA but no protein. It was complementary 3' uts of lin14. Lin 4 developmentally regulates lin 14 during development. It hybridizes to the 3' untranslated region. It targest lin 28 too. You have a little RNA that is somehow repressing expression of two unrelated gens. It's only turned on at one stage. See if there were any endogenous RNAi. Do they make RNAs themselves He took RNA and tagged it and PCR and made DNA cDNA and sequenced a whole library fo these. He found, he looked at where these were transcribed in Northerns. They're transcribed in 0 to 3 hours and so on. these RNAs are developmentally regulated themselves. He did some folding and it turns out they're clustered. All these genes he cloned are clustered and they form big long hairpins. The RNA he was after was in red. Ecach one of these had a single or 2 of these. He called these microRNAs becaue they camefrom the genome. miRNAs have a different property of hybridization. If you take an siRNA, it will make a perferct duplex. If you take micro RNA, you get bulges and mismatches. It turns out if you get p3erfect matching, it goes to degradation. If it's important you get microRNA. Here's lin4 against lin14. Here's the original large product. They found the second enzyme Drosha and lacked the helicase. They found that drosha was actually responsible for cutting these hup and making pri mi rna. Pasha binds and tell Drosha where to cut. Pasha is also called DGCR8. It cuts and produces this bottom peiece. Drosha makes a pre-miRNA and it makes a hairpin. the miRNA is in one of these strands. This is just a digagram of what they think is happening in the nucleus. You have these large transcripts where Pasha and Drosha bind and produce the pre-miRNA which is about 70nt. They're mismatches and bulges. This mature miRNA actually goes to the same complex that dsRNA will go to and Dicer comes in and now dicer recgonizes this and cuts out the miRNA with two overhanging nt. They look just like siRNAs but they have bulges. Much more prevalent in the genome and they're really important. These miRNAs would bind to argonaut. What do they do? You have pOLII, PrimiRNA that is regulated by drosha and pasha, then you ge premirna and dicer cuts out the exact mirna and the other strand gets translated. If it's target is 3' of a mRNA, it will preepress translation of that message. They specifically repress translation of specific genes. You've got all these genes that has nothing to dowith the gene but they're from the gene and they get transcribed tranlstate, whene it's done, it will hybridize incompletely and it inhibits translation. mRNA gets degraded sponataneoulsy. Here's a dsRNA exogenous ly put in and gets in This just repeats what I said. it's cut by dicer and that goes on to regulate translation. Another class of little RNAs was discovered by this method of clongin. These were 26nt instead of 21 or 22. They're called piwi RNA. They're involved in degradation of transposon mRNA and they destruct transposable elements during germline. That's their purpose by going through RNA interference. Here's dicer, What els des miRNAs do Turns out that miRNAs, foreign dsRNA gets itno the cell somehow. miRNAs now, let's say you havve a transgene that comes in, They silence transctiption of chromatin. By methylating a histone. these miRNA can turn on silencing of chromatin in the nucleus and that's by methilation of the histone. These are the guiding things for other enzymes. Therefore we have a nother role of miRNA which is heterochromatin fomration. This has led to a new way of looking at the genome. it's not nearly paradigm shift. We have a protein encoding gene. wtih a promoter. Now we know there are a lto of little RNAs calle dmiRNA that 30% of your genome is actually miRNA. miRNAs can base pair incompletely with product gene and regulate it during development. The whole new concept is a lot of the regulation that we observe is caused by miRNAs. Thesee did things like regualte translation, silenceing, heterochromatin, etc. chronology. What is it for? How ancient? If you eliminate RNAi you get incrase transposable element.s htat's wone possible role. it's in T. brucei and id divereged from the main eukarytoe linatea long time ago. RNA i had evovled in a very early euk cell. Argonaute has a relationship to viral RT, transposases, and integraseiRNAs mya have been primers for the replication of early genomes. This is pure speculation. Piwi is separatge Portential therapeutic uses. How do we get the siRNAs in? How do you ge the the siRNA in the cell? Viral vectors, gene theerapy to express specific siRNA but gene therapy has its own problems. What companies have done is make syunthetic siRNA. It's not recognized by nucelases a... turnover is much slower. You can make siRNa capable of penetrating cells, hydrophobic. A big problem with humans is that if you put a logn dsRNA into an org, it responds by making animmune repsonse. If you use siRNAs or hairpiins, the mammalian cells don't seem to make immuen response to those. Mouse model have had good results with autoimmune hepaitis. Why is pulmonary infection a good way? You can breathe it in. Retina too. It's closed system and you get good effects. hey've knocked down HIV in models with siRNA. ...
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- Fall '08