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
