Mechanistic aspects and therapeutic applications of RNAi

Published on October 1, 2007 Reviewed on August 21, 2015   44 min

Other Talks in the Category: Cell Biology

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My name is John Rossi, and I'm from the Division of Molecular Biology at the Beckman Research Institute of the City of Hope in Duarte, California. I am going to be talking about the Mechanistic Aspects and Therapeutic Applications of RNAi, with specific emphasis on understanding some of the mechanisms aspect when we apply RNAi to the treatment of disease such as infectious diseases like HIV. We'll have a full understanding of what we are dealing with in terms of the cellular mechanisms. This first slide depicts rather busy scenario of
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the many different ways that small RNAs can regulate gene expression in eukaryotic cells. And I want to start on the left hand part of the slide by showing the endogenous microRNA pathway. As we are pretty well familiar with by now, the largest role that RNAi plays in mammalian cells is through the microRNA pathway. And this involves the expression usually by RNA polymerase 2 promoters of primary hairpin transcripts, often in clusters that are processed by a nuclear enzyme with an RNAse 2 family member called Drosha into pre-microRNAs, which are still in the form of the hairpin usually having complete Watson-Crick base pairing in their stems. These pre-microRNAs are exported through the cytoplasm via a carrier called Exportin5, again, processed by the RNAse three family member Dicer which is in complex with these two other cellular factors, a protein called the TAR and a binding protein and another one called PACT. This complex then processes these RNAs and hands it off to components of the RNA induced silencing complex, primarily the Argonaute proteins. Once the antisense strand is selected and in the case of microRNA it's not really clear how it's selected, it is aligned then with partially complementary sequences in the three prime untranslated region of target RNAs, wherein, these complexes will block translational initiation. The other arm in this pathway is the siRNA or small interfering RNA arm, and that involves processing or introduction of the cells of perfectly matched duplex RNAs that are either going to be substrates for the enzyme Dicer and its associated components, or these siRNAs can be introduced preformed as 19 based duplexes with two base three prime overhangs. Now, in the case of siRNAs, the mechanism for selection of the antisense strand is actually better understood. And that involves the thermodynamic stability of the ends of these molecules. So the less thermodynamically stable end is the one that's chosen for entry into the binding site of Argonaute two. And the passenger strand is actually cleaved in the Argonaute two complex and eliminated, thereby leaving the antisense strand to guide the targeted destruction of messenger RNAs. These two pathways are completely interchangeable and it's all based upon the extent of base pairing and where that base pairing takes place. So siRNAs can actually be microRNAs in terms of translational inhibition, if they have partial complementarity to the target sequence in the three prime translated region. And same is true for microRNAs which can actually act as siRNAs, if they have perfect complementarity to the target region. The other pathway that is shown here is the chromatin silencing pathway. I won't be dwelling on this, but I just want the viewers to be aware the fact that small RNAs can, in fact, direct histone methylation. Perhaps, DNA methylation in the long term resulting in epigenetic silencing of chromatin, in particular, heterochromatic regions around centromeres may be targets for RNAi base silencing. And I do also want to emphasize that, since the native pathway is primarily the microRNA pathway in mammalian cells, that when we express or introduce foreign siRNAs into these cells, we are competing with various steps of that pathway. And this is something that needs to be taken into consideration in designing experiments especially for therapeutic applications. This slide just illustrates the various ways
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Mechanistic aspects and therapeutic applications of RNAi

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