RNA Interference Flashcards
1
Q
What is RNAi?
A
RNA interference
2
Q
What is dsRNA? (2)
A
- Double stranded RNA
- Longer than 30 nucleotides
3
Q
What is miRNA? (5)
A
- microRNA
- 21-25nt
- Encoded by endogenous genes
- Hairpin precursors ~70nt
- Recognise multiple mRNA targets
4
Q
What is siRNA? (5)
A
- Short-interfering RNA
- 21-25nt
- Mostly exogenous origin (mainly experimental tool to knockdown protein expression)
- dsRNA precursors
- May be target specific
5
Q
What is post-transcriptional gene silencing (PTGS)/quelling? (3)
A
- Jorgenson attempted to overexpress chalcone synthase protein in petunias to make them deeper purple but ended up with some areas with no chalcone synthase expression instead (co-suppression of both endogenous and introduced genes)
- Called it PGTS
- Similar results seen in N.crassa (called it quelling)
6
Q
What did Fire and Mello first discover? (4)
A
- Antisense RNAs were used to bind to and silence target RNAs by preventing translation or triggering degradation before RNAi took off
- Fire and Mello found that both sense and antisense RNAs are sufficient for silencing of the endogenous gene = strange
- Silencing persisted even though the injected RNA was easily degraded
- RNA for silencing was often generated using a template DNA strand and a T7 bacteriophage polymerase which can make ectopic transcripts because it jumps to the other DNA strand and carries on transcribing = low level of double stranded RNA present
7
Q
What experiment led to the discovery of RNAi? (6)
A
- Unc-22 gene inactivation in C.elegans causes uncoordinated twitching
- Injected (ss) sense RNA, antisense or both (ds) into C.elegans gut
- dsRNA was orders of magnitude more effective than ssRNA, only a few molecules needed
- Unc-22 null phenotype was also in the progeny of injected worms
- Inactivation was due to degradation of target mRNA
- Coined the term “RNA interference” (Fire et al., 1998)
8
Q
How was siRNA identified? (2)
A
- Long dsRNA gets processed into duplexes 21-25bp long in plants (Baulcombe)
- Also happens in drosophila (Elbashir) and the fragments are siRNAs which are necessary for degradation of the target RNA
9
Q
How are miRNAs made? (6)
A
- Transcription in the nucleus produces a pri-microRNA which contains a hairpin sequence which is the microRNA sequence
- Microprocessor (contains DGCR8 and Drosha) removes flanking sequences resulting in a smaller hairpin structure = pre-microRNA
- Pre-microRNA exported from the nucleus to cytoplasm
- Hairpin loop chopped off by Dicer
- AGO protein selects the microRNA strand that is complementary to the target and delivers it to the target
- microRNA causes cleavage/degradation or translational suppression of the target (or both)
10
Q
What is an example of microRNA?
A
Let-7
11
Q
What are mirtrons? (2)
A
- A type of microRNA produced from introns of protein-encoding mRNA primary transcripts
- Produced by splicing
12
Q
What are the components of the microprocessor? (2)
A
- DGCR8
- Drosha
13
Q
What is Drosha?
A
RNase III type enzyme
14
Q
How does the microprocessor know where to cleave? (2)
A
- DGCR8 positions binds to the loop sequence and positions Drosha
- Drosha cleaves the pri-miRNA ~11bp up the stem producing pre-miRNA
15
Q
How are pre-miRNAs exported to the cytoplasm? (2)
A
- Exportin5 and Ran-GTP GTPase binds to pre-miRNA, complex escorts pre-miRNA to the cytoplasm
- GTP hydrolysis when in the cytoplasm induced by RanGAP on the cytoplasmic side of the nuclear pore which drives disassembly of the complex and releases pre-miRNA for Dicer processing
16
Q
What does Dicer do? (3)
A
- Phosphorylates 5’ end of synthetic duplexes
- Chops up long dsRNA into 21-25nt duplexes
- Main job is to chop off the hairpin loop sequence leaving double stranded short duplex to be used
17
Q
How does the argonaute (AGO) complex know which strand to select? (3)
A
- There is thermodynamic asymmetry in the duplex and AGO will unwind from the end that is the easiest (least number of hydrogen bonds, CG has 3 so strongest, AU is weaker and GU wobble bp is also weak)
- The 5’ strand at the side being unwound is the guide strand, other one is the passenger strand
- AGO complex cleaves the passenger strand and associates with the guide strand = RISC
18
Q
What is RISC? (2)
A
- RNA induced silencing complex
- Guide strand bound to AGO complex
19
Q
What are the different ways in which the RISC complex suppresses gene expression? (6)
A
- RISC microRNA complex binding site is usually on 3’ UTR of target mRNA
- Most commonly RISC cleaves the target mRNA and it is degraded
- Block translation initiation by inhibiting initiation factors or block elongation
- Drive deadenylation by recruiting CCR4NOT complex, mRNA susceptible to degradation without polyA
- Proteolysis of the nascent polypeptide as it is being translated
- (Cleave and degrade or translational repression)
19
Q
What is the interferon response? (3)
A
- Long dsRNA injected into mammalian cell triggers production of interferons (anti-viral proteins which cause an inflammatory response) so useless as a tool
- Cascade triggers activation of RNase L (non-specific ribonuclease) which degrades all RNA in the cell
- PKR enzyme autophosphorylates (active) and phosphorylates EIF2alpha (translation initiation factor) so it can’t be recycled for further rounds of translation initiation (blocks protein synthesis)
20
Q
What can be used in mammalian cells instead of dsRNA?
A
siRNAs which are too short to trigger interferon response
21
Q
What does RNAi achieve?
A
KnockDOWN of target gene (just reducing the levels of target mRNA), not knockout
22
Q
How can you make a miRNA mimic in vivo instead of buying siRNAs? (4)
A
- Expression of hairpin like structure from a plasmid that will be processed by Drosha and produce a miRNA mimic
- Make the 2 strands of the hairpin homologous to the target gene
- Useful for silencing of essential proteins to identify function, inducible gene silencing
- Can make short hairpin RNA (shRNA) (looks very similar to miRNA)
23
Q
What was the primary method for reducing gene function before RNAi? (2)
A
- Mouse knockouts
- Expensive and more difficult
24
How was RNAi used for proteasome screening? (4)
- Used a range of shRNAs to knockdown genes and identify the subunits of the proteasome
- Used GFP fused to PEST sequence as the reporter
- PEST sequence targets to proteasome for degradation so the GFP is very unstable
- Knockdown of proteasome subunit genes stops degradation of GFP
25
How does RNAi by feeding in agar plates work? (4)
- Make E.coli expressing dsRNA of target gene
- Add C.elegans eggs to the plate
- Worms eat the bacteria, goes into gut, releases dsRNA into the worm which can do RNAi
- Make a library of bacteria to do a genome wide screen of C.elegans
26
What is an example of an RNAi screen? (2)
- C.elegans screen to identify genes which drive fat deposition
- Daf2 mutation causes fat worm phenotype
- Bacteria library screen knockdown to identify genes which cause fat deposition e.g. HMG-CoA reductase
