RNA Interference Flashcards
What is RNAi?
RNA interference
What is dsRNA? (2)
- Double stranded RNA
- Longer than 30 nucleotides
What is miRNA? (5)
- microRNA
- 21-25nt
- Encoded by endogenous genes
- Hairpin precursors ~70nt
- Recognise multiple mRNA targets
What is siRNA? (5)
- Short-interfering RNA
- 21-25nt
- Mostly exogenous origin (mainly experimental tool to knockdown protein expression)
- dsRNA precursors
- May be target specific
What is post-transcriptional gene silencing (PTGS)/quelling? (3)
- 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)
What did Fire and Mello first discover? (4)
- 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
What experiment led to the discovery of RNAi? (6)
- 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)
How was siRNA identified? (2)
- 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
How are miRNAs made? (6)
- 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)
What is an example of microRNA?
Let-7
What are mirtrons? (2)
- A type of microRNA produced from introns of protein-encoding mRNA primary transcripts
- Produced by splicing
What are the components of the microprocessor? (2)
- DGCR8
- Drosha
What is Drosha?
RNase III type enzyme
How does the microprocessor know where to cleave? (2)
- DGCR8 positions binds to the loop sequence and positions Drosha
- Drosha cleaves the pri-miRNA ~11bp up the stem producing pre-miRNA
How are pre-miRNAs exported to the cytoplasm? (2)
- 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
What does Dicer do? (3)
- 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
How does the argonaute (AGO) complex know which strand to select? (3)
- 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
What is RISC? (2)
- RNA induced silencing complex
- Guide strand bound to AGO complex
What are the different ways in which the RISC complex suppresses gene expression? (6)
- 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)
What is the interferon response? (3)
- 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)
What can be used in mammalian cells instead of dsRNA?
siRNAs which are too short to trigger interferon response
What does RNAi achieve?
KnockDOWN of target gene (just reducing the levels of target mRNA), not knockout
How can you make a miRNA mimic in vivo instead of buying siRNAs? (4)
- 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)
What was the primary method for reducing gene function before RNAi? (2)
- Mouse knockouts
- Expensive and more difficult
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
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
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