RNA Interference

by Kevin Ahern, PhD

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    00:00 Now another important process recently discovered that all RNAs also function in, is a very critical and interesting process called RNA interference.

    00:10 Now RNA interference is a process that's stimulated by the presence of double stranded RNA inside of cell.

    00:19 Now as we are gonna see this can happen either as a result of the cell making double stranded RNA or by a virus that is invaded.

    00:26 And the invasion of a virus bringing in double stranded RNA is sure cue for a signal for a problem.

    00:34 Now the two different forms of double stranded RNA that can exists in the cell are known as micro RNAs or miRNAs and these have cellular origins.

    00:43 The siRNAs or silencing RNAs come from an external source like a virus for example.

    00:49 Now I note that biotechnologist are also using double stranded RNAs as a means of controlling genes for biotechnology purposes.

    00:59 So that's another way that foreign siRNA can get into cells.

    01:05 Now the process is quite wide spread in eukaryotic cells.

    01:08 It occurs in plants. It occurs in animals and it plays a wide variety of roles.

    01:15 The actions performed in this process are called RNA interference or RNAi.

    01:20 And what these double stranded RNA molecules result in is it the interfering of the translation of targeted genes.

    01:31 Now the ability to use this technology to target and specifically stop the production of certain proteins allows a researcher to do incredible things. But more importantly it allows the cell that has this to both protected cells from invaders and also to control its own gene expression.

    01:51 RNA interference operates through the silencing of gene expression and that silencing, as I said, is interference in the way that proteins are made.

    02:01 Now this occurs as a result of, first of all, the appearance in the cell of a double stranded RNA and there is an enzyme called dicer.

    02:12 And just like a dicer that you might have in a kitchen, this dicer's job is to take that double stranded RNA and chop it into bite size chunks and those bite size chunks are about 20 base pairs long, as we shall see.

    02:25 So these 20 base pairs chunks at this point are called silencing RNAs if they came from an external source and micro RNAs if they came from a cellular source.

    02:37 These pieces of RNA can then be bound by the RNA induced silencing complex or what is knows as RISC. Now I am gonna show you this whole process in just a minute.

    02:48 Now this figure illustrates the process that I was just describing in words on the previous slide.

    02:54 We can see actually two things occurring right here.

    02:55 In the process occurring on the right, where I am describing a cellular process that starts with the production of an RNA that makes a double stranded RNA used to create the miRNAs that I described.

    03:08 In the process on the left we have simply the dsRNA that is appeared in the cell by the foreign source whether it's a virus or is by a researcher placing that within there.

    03:21 I am gonna start with the process on the right to describe yet.

    03:24 The cell has encoded within its genome certain sequences that when transcribed produce structure like you can see on the right.

    03:33 That double stranded structure with tails hanging off of it and the poly A that you see on it is called a pri-miRNA.

    03:40 The name doesn't really matter. But that pri-miRNA gets processed to make what will ultimately become the miRNA.

    03:49 So there is an enzyme called drosha that cuts some of the ins off of the pri-miRNA and creates the smaller structure that you see on the image.

    04:02 The pri-miRNA is then moved out of the nucleus, as you can see, where it is then attached to the enzyme known as dicer.

    04:10 Now at this point the two processes the siRNA and the miRNA become the same.

    04:17 What dicer does is it takes that double stranded pri-miRNA or the double stranded RNA that you see on the left from the foreign source and chops them into the 20 base pairs sequences that I have described.

    04:33 You can see for example a perfect duplex on the siRNA side and sort of a mismatched duplex with a bulge on the miRNA side.

    04:43 That's very common for miRNAs.

    04:47 Dicer after chopping this into 20 nucleotide blocks then peels away one of the strands.

    04:56 And the peeling away of that one strand leaves a single stranded siRNA or a single stranded miRNA that is then grabbed by the RISC. Now the RISC takes that individual sequence and carries it to a messenger RNA.

    05:14 Now the significance of the fact that there is a single strand at this point is due to the fact that this single strand would be complimentary to a target messenger RNA, as we shall see.

    05:27 Now after the RISC has complexed with that single stranded RNA whether it was an miRNA or whether it was an siRNA at this point doesn't matter.

    05:35 That RISC RNA complex then goes and seeks messenger RNA. Messenger RNAs, of course, are coding for individual proteins.

    05:46 If RISC finds a sequence that's complementary to the RNA that it is carrying it aligns that sequence with the specific region in the messenger RNA as you can see here.

    05:56 And then an enzymatic activity in the RISC complex called argonaute actually cleaves the target messenger RNA, as you can see here.

    06:07 Now that cleaving of the target messenger RNA means that you have destroyed the coding for a protein.

    06:14 In this way this protein that was coded by this messenger RNA can no longer be made.

    06:20 Well, this has a couple of implication as you could imagine.

    06:24 This has very obvious protection effects for the cell against an invading virus.

    06:31 If the invading virus makes a double stranded RNA in the process of its life cycle then this siRNA system will stop the production of targeted virus proteins.

    06:44 Now also it may seem a little odd but the miRNA also plays a role in regulating gene expression; because, the miRNA is actually stopping in this case production of a cellular gene that would otherwise make this protein. And it might seem as very inefficient for this cell to make a messenger RNA and then degrade the messenger RNA.

    07:09 But that probably makes more sense than continuing to make a protein that the cell wouldn't otherwise need.

    07:15 So this miRNA system allows for an additional level of protection or an additional level of control of a cellular gene expression.

    07:25 In any event, what happens here is that the translation of the messenger RNA stopped.

    07:29 Now I showed mention the fact that this processing that happens doesn't have to cut the RNA.

    07:37 It can also involve a simple binding of that miRNA or siRNA sequence to the messenger RNA and stop translation by the formation of that duplex alone.

    About the Lecture

    The lecture RNA Interference by Kevin Ahern, PhD is from the course RNA and the Genetic Code.

    Included Quiz Questions

    1. It relies on formation of duplex RNA sequences in cells
    2. It occurs when transcription of a gene is blocked
    3. It involves pairing of a target RNA to a complementary sequence by a protein called dicer
    4. It arises when the RISC protein destroys an RNA duplex
    1. Dicer
    2. RNAse
    3. DNAse
    4. Protease
    5. Ligase
    1. The siRNA contains a mismatched duplex with a bulge on it, whereas the miRNA represents a perfect duplex system
    2. The presence of double-stranded RNA molecules in the eukaryotic cells stimulates the RNAi response
    3. The RNAi response helps the cell to protect itself from the invader as well as to control its own gene expressions
    4. The double-stranded viral RNA gets chopped off by enzyme called dicer into silencing RNA molecules of 20 basepair lengths
    5. Drosha and Dicer are two enzymes, which participate in the RNA interference process in the eukaryotic cells
    1. Argonaute, due to which it cleaves the target mRNA
    2. Dicer, due to which it cleaves the pri-miRNA
    3. Drosha, due to which it cleaves the pri-siRNA
    4. Drosha-dicer, due to which it cleaves the pri-viral RNA
    5. Lyase, due to which it cleaves the pri-mRNA

    Author of lecture RNA Interference

     Kevin Ahern, PhD

    Kevin Ahern, PhD

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