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What did Andrew Fire and Craig Mello recieve a Nobel Prize for in 2006?
Their discovery of RNA interference - gene silencing by dsRNA.
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What did Andrew Fire and Craig Mello observe (to their bewilderment) in their research?
That Double Stranded RNA was more effective than Single Stranded RNA at interfering.
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What was the initial theory surrounding RNA interference?
That Antisense RNA binds to it's complimentary region and prevents translation.
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What was a key argument against the initial hypothesis?
That both Antisense and Sense RNA could cause interference.
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What Organism did Fire and Mello use to test the effects of RNA interference?
C. elegens gonads.
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What mRNA did Fire and Mello use to determine the effects of RNA interference?
How did they visualize it?
- They used mex-3 mRNA.
- They Visualized it using a labelled probe.
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What was the effect of single stranded RNA on Mex-3?
A significantly reduced amount of Mex-3, but still a reasonable amount present.
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What was the effect of dsRNA on Mex-3 mRNA?
No visible Mex-3 at all.
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What type of Hybridization did Fire and Mello use?
In Situ Hybridization?
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What is an In Situ hybridization?
- When the test is done in the organism.
- "Probe added to tissue".
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What was the effect of adding RNA to one cell in a tissue?
The effects extended (somewhat evenly) to all cells.
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What is Dicer?!
- A member of the RNAase III family.
- A Double-Strand-Specific Endonuclease.
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What does Dicer do?
Dicer cleaves trigger dsRNA into 21-23 dsRNA fragments.
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What is the substrate for Dicer?
(Full Length) Trigger dsRNA.
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How are siRNA's made?
Dicer cleaves ds Trigger RNA which creates 21-23nt fragments known as siRNA.
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What does siRNA stand for?
Small Interfering RNA.
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What is "special" about siRNA's?
- They are small
- They are Double Stranded
- They have "Sticky ends"
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What kind of overhand does Dicer produce?
Dicer-produced siRNA's have sticky ends with a 2nt 3' overhang.
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What does RISC stand for?
RNA-Induced silencing complex.
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Briefly describe how RISC acts.
- siRNA complexes with RISC.
- The RISC complex binds to complimentary RNA.
- Cleavage occurs
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RISC contains an RNAse. Is it the same as dicer?
No, it is Slicer.
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Which comes first, Slicer of Dicer?
Dicer (Formation of siRNA) then Slicer (RISC).
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What is microRNA?
- Small RNA's produced naturally
- They act by RNA interference
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How are miRNA's produced?
- RNA transcripts form stem loops
- Pasha and Drosha cleave the stem loop
- The Stem loop leaves the nucleus
- The Loop element is cleaved
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What are some properties of miRNA's?
- Very similar to siRNA's
- Short sequence
- 2nt 3' Overhang
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What is the role of Drosha in RNA interference?
Drosha cleaves the stem loop at the bottom, creating the first sticky end.
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Where is Drosha found?
In the nucleus (Nucleus Localized)
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What kind of Enzyme removes the loop element after Nuclear Export?
Dicer
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In which two ways can miRNA's interfere?
- Near-perfect base pairing interactions
- Imperfect base pairing interactions (Bulges)
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How does imperfect pairing of miRNA prevent gene expression?
The bulge created by poor base pairing creates a "roadblock" in translation.
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How does Near-Perfect pairing of miRNA prevent Gene Expression?
The RISC complex will cleave the target RNA using slicer.
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Which cleaving miRNA mechanism(s) do animals use?
- Perfect Pairing
- Imperfect Pairing
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Which cleaving Mechanism(s) do plants use?
Perfect Pairing.
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What is the Argonaute Protein?
An nuclease similar to RNAase H.
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What is the substrate for RNAase H?
RNA-DNA hybrids.
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What is the role of RNAase H?
Argonaute specifically cuts RNA in an RNA-DNA hybrid.
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What was the first indication that argonaute has enzymatic activity?
It has a similar structure to RNAase H.
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How does Argonaute accommodate it's substrate?
It has a cleft allowing for insertion of dsRNA.
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What is the sequence of Domains in Argonaute?
N, PAZ, MID, PIWI
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How was Argonaute's enzymatic activity proven?
In vitro demonstrations showed that just Argonaute+siRNA was required to cleave a larger mRNA.
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Using electrophoresis, how can you tell where Argonaute is cutting?
- Determine the size of the fragments.
- Electrophoresis of an Argonaute+siRNA system will yield three marks; Substrate, 5' Product & 3' Product.
- By determining the relative sizes of the 5' and 3' products it is possible to decipher the exact cutting location.
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Is Argonaute the slicer in RISC? How was this initially determined?
- Yes.
- It was determined by an In Vitro Assay.
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How does dsRNA associate with Argonaute?
The 3' end binds to the PAZ domain
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How does positioning of the 3' of dsRNA affect cleaving?
Positioning of the 3' end of siRNA (siRNA-RNA hybrid) in the PAZ domain lines the cleavage site up with the PIWI domain which cleaves.
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How does interference by dsRNA work?
- dsRNA is cleaved by Dicer producing siRNA
- SiRNA is loaded into Risc
- The Risc Complex cleaved complimentary mRNA
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How was the In Vitro Assay set up?
- The Argonaute Gene was Cloned, Expressed and Purified.
- RNA was labelled and added
- Results were collected
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What is the R2D2 protein?
A protein used in the formation of RISC.
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How is the RISC Complex B formed?
- siRNA binds to Dicer and R2D2 asymmetrically.
- The less stable helical end binds to Dicer
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How is the strand from double standed siRNA chosen in Complex B?
The 5' end bound to Dicer is used in the later RISC complex.
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What are the two proteins in Complex B? (RISC formation)
Dicer and R2D2
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How/when is the RISC loading complex formed?
- After Complex B.
- Additional proteins are added to R2D2 and Slicer, the passenger strand (5' bound to R2D2) is cut and removed.
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What is the order of Complexes in RISC formation?
Complex -> RLC -> RISC
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What is the molecule in cells responsible for Site-Specific Cleavage?
RISC
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What is Transitive RNAi?
When a small amount of limited dsRNA's can interfere with gene expression over a Much larger range.
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What is RdRp?
RNA-directed RNA polymerase.
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How does Transitive RNAi use RdRp?
- Some siRNA will not enter the RISC complex but will bind independently to mRNA.
- RdRp transcribes an RNA product complimentary to the large mRNA strand.
- The nascent dsRNA is then cleaved by dicer, creating further (more varied) siRNA's.
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