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Printable Handouts
Navigable Slide Index
- Introduction
- Multiple pathways of RNAi
- Triggering RNAi in mammalian cells
- Is RNAi coupled with translation?
- Iron regulated translational switch
- Iron-regulated RNAi - methods
- Iron-regulated RNAi - results
- Iron-regulated RNAi - summary of the study
- RNAi takes place in cytoplasmic P bodies
- Possible mechanisms of P body formation
- Dicer sunstrate RNAs enhance RNAi potency
- Potential interactions of TRBP and Dicer
- Optimizing synthetic siRNA efficacy
- RNAs incubated with recombinant human Dicer
- Recombinant Dicer digestion of RNA duplexes
- Mass Spec of Dicer products
- Longer siRNA are more effective
- Dicer substrate has higher RNAi efficacy
- No potent siRNAs derive from the 27mer
- In vitro dicing of 27-mer does not improve efficacy
- Longer siRNa are effective for longer period of time
- 27mer RNAi potency in cleavaging of La
- Directing the 21mer RNA produced from 27mers
- TRBP-Dicer complex
- EGFP site 2
- The R-form is the most effective
- L-form enhances use of sense strand
- How is antisense strand selected?
- Antisense strand selection
- 5' LNA modified siRNA
- LNA at +2 position of the sense strand works best
- Conclusions - strand specificity
- RNA interference as a potential HIV therapy
- Hematopoiesis
- T cell-based gene therapy
- Combinatorial RNA-based therapy for HIV
- HIV life cycle
- Targeting early transcripts (tat and rev)
- Targeting early transcripts - methods
- Lentiviral vector based upon HIV7
- shRNAs produced from triple hairpin transcription
- Triple hairpin approach is very effective
- Resistant mutants don't arise against triple hairpin
- Analysis of the mutants (1)
- Analysis of the mutants (2)
- Conclusions - triple hairpin approach
- Tat-inducible system
- HIV-1-inducible siRNA system - mechanism
- Tat inducibility
- Lentiviral packaging system
- Lentiviral system with open reading frame
- LTR vector with huRevM11
- LTR vector with huRevM10 protects against HIV
- LTR vector with huRevM10 protection of CEM cells
- No shRNA resistant mutants arise after 70 days
- Conclusion - anti-Rev shRNA
- Acknowledgements
Topics Covered
- RNAi mechanisms
- RNAi and HIV infection
- Lentiviral vector-mediated transduction of stem cells and T-cells
- Gene therapy based treatment of HIV-1
- HIV mutants that abrogate RNAi
- Multiplexing different siRNAs and siRNAs with other antiviral RNAs to minimize viral resistance
- An HIV Tat inducible shRNA expression system
- Multiplexing shRNA with dominant negative M10 protein inhibition of HIV
Links
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Talk Citation
Rossi, J. (2015, August 21). Mechanistic aspects and therapeutic applications of RNAi [Video file]. In The Biomedical & Life Sciences Collection, Henry Stewart Talks. Retrieved October 4, 2024, from https://doi.org/10.69645/TIQD2320.Export Citation (RIS)
Publication History
Financial Disclosures
- Professor John Rossi is a cofounder of MiNA Therapeutics.
Mechanistic aspects and therapeutic applications of RNAi
A selection of talks on Cell Biology
Transcript
Please wait while the transcript is being prepared...
0:00
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.
0:26
This first slide depicts rather busy scenario of
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