Bio 3000 - RNA Interference
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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.
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.
What was the initial theory surrounding RNA interference?
That Antisense RNA binds to it's complimentary region and prevents translation.
What was a key argument against the initial hypothesis?
That both Antisense and Sense RNA could cause interference.
What Organism did Fire and Mello use to test the effects of RNA interference?
C. elegens gonads.
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.
What was the effect of single stranded RNA on Mex-3?
A significantly reduced amount of Mex-3, but still a reasonable amount present.
What was the effect of dsRNA on Mex-3 mRNA?
No visible Mex-3 at all.
What type of Hybridization did Fire and Mello use?
In Situ Hybridization?
What is an In Situ hybridization?
- When the test is done in the organism.
- "Probe added to tissue".
What was the effect of adding RNA to one cell in a tissue?
The effects extended (somewhat evenly) to all cells.
What is Dicer?!
- A member of the RNAase III family.
- A Double-Strand-Specific Endonuclease.
What does Dicer do?
Dicer cleaves trigger dsRNA into 21-23 dsRNA fragments.
What is the substrate for Dicer?
(Full Length) Trigger dsRNA.
How are siRNA's made?
Dicer cleaves ds Trigger RNA which creates 21-23nt fragments known as siRNA.
What does siRNA stand for?
Small Interfering RNA.
What is "special" about siRNA's?
- They are small
- They are Double Stranded
- They have "Sticky ends"
What kind of overhand does Dicer produce?
Dicer-produced siRNA's have sticky ends with a 2nt 3' overhang.
What does RISC stand for?
RNA-Induced silencing complex.
Briefly describe how RISC acts.
- siRNA complexes with RISC.
- The RISC complex binds to complimentary RNA.
- Cleavage occurs
RISC contains an RNAse. Is it the same as dicer?
No, it is Slicer.
Which comes first, Slicer of Dicer?
Dicer (Formation of siRNA) then Slicer (RISC).
What is microRNA?
- Small RNA's produced naturally
- They act by RNA interference
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
What are some properties of miRNA's?
- Very similar to siRNA's
- Short sequence
- 2nt 3' Overhang
What is the role of Drosha in RNA interference?
Drosha cleaves the stem loop at the bottom, creating the first sticky end.
Where is Drosha found?
In the nucleus (Nucleus Localized)
What kind of Enzyme removes the loop element after Nuclear Export?
In which two ways can miRNA's interfere?
- Near-perfect base pairing interactions
- Imperfect base pairing interactions (Bulges)
How does imperfect pairing of miRNA prevent gene expression?
The bulge created by poor base pairing creates a "roadblock" in translation.
How does Near-Perfect pairing of miRNA prevent Gene Expression?
The RISC complex will cleave the target RNA using slicer.
Which cleaving miRNA mechanism(s) do animals use?
- Perfect Pairing
- Imperfect Pairing
Which cleaving Mechanism(s) do plants use?
What is the Argonaute Protein?
An nuclease similar to RNAase H.
What is the substrate for RNAase H?
What is the role of RNAase H?
Argonaute specifically cuts RNA in an RNA-DNA hybrid.
What was the first indication that argonaute has enzymatic activity?
It has a similar structure to RNAase H.
How does Argonaute accommodate it's substrate?
It has a cleft allowing for insertion of dsRNA.
What is the sequence of Domains in Argonaute?
N, PAZ, MID, PIWI
How was Argonaute's enzymatic activity proven?
In vitro demonstrations showed that just Argonaute+siRNA was required to cleave a larger mRNA.
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.
Is Argonaute the slicer in RISC? How was this initially determined?
- It was determined by an In Vitro Assay.
How does dsRNA associate with Argonaute?
The 3' end binds to the PAZ domain
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.
How does interference by dsRNA work?
- dsRNA is cleaved by Dicer producing siRNA
- SiRNA is loaded into Risc
- The Risc Complex cleaved complimentary mRNA
How was the In Vitro Assay set up?
- The Argonaute Gene was Cloned, Expressed and Purified.
- RNA was labelled and added
- Results were collected
What is the R2D2 protein?
A protein used in the formation of RISC.
How is the RISC Complex B formed?
- siRNA binds to Dicer and R2D2 asymmetrically.
- The less stable helical end binds to Dicer
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.
What are the two proteins in Complex B? (RISC formation)
Dicer and R2D2
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.
What is the order of Complexes in RISC formation?
Complex -> RLC -> RISC
What is the molecule in cells responsible for Site-Specific Cleavage?
What is Transitive RNAi?
When a small amount of limited dsRNA's can interfere with gene expression over a Much larger range.
What is RdRp?
RNA-directed RNA polymerase.
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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