4 - RNA Interference - Wilson

Question 1

give the definitions for 
- dsRNA
- miRNA
- siRNA 
(for miRNA state their precursors and targets and origins)

Answer 1

dsRNA; RNA held in a duplex longer than 30nt 
miRNA; micro-RNA
- 21-25nt 
- derived from endogenous genes 
- hairpin precursors (around 70nt) 
- recognise multiple targets 
siRNA; short interfering RNA
- exogenous origin 
- dsRNA precursors 
- recognise specific targets but can have off-target effects

Question 2

describe the experiment that discovered this RNA interference

Answer 2

• experiment in C. elegans Unc-22 gene
• designed mRNAs against the Unc-22 gene. null phenotype -> uncoordinated twitching
• sense, antisense or both was injected into C. elegant gut
• dsRNA was orders more effective than ssRNA
• the Unc-22 null phenotype was also seen in progeny of injected worms
• inactivation was due to the degradation of the target mRNA

Question 3

what does q3 325-4 word show?

Answer 3

• injection of dsRNA that recognises GFP selectively prevents expression of the GTP protein in both larval and adult stages of the worm

Question 4

give the first 2 types of siRNA identified

Answer 4

• plants showing co-suppression; 21-25nt dsRNA. not found in other plants and their sequence similar to target gene being suppressed
• Drosophila; long dsRNA processed into 25bp fragments. fragments = siRNA

Question 5

draw an OVERALL diagram of the biogenesis of microRNA and its assembly into microribonucleoprotein complexes (miRNPs)

Answer 5

325 - 4 word

Question 6

describe in detail the cleavage of the pri-mRNA complex and DAG

Answer 6

• pri-mRNA processing by the microprocessor complex (Drosha and DGCR8) into pre-mRNA that can then be exported
• cleavage occurs around 11bp up the stem
• DGCR8 binds as a dimer to the dsRNA hairpin and directs localisation of Drosha to correct site for cleavage
• Drosha = RNase III type enzyme
• cleavage of the pri-mRNA leaves a 3’ overhang

Question 7

describe the export of pre-miRNAs to the cytoplasm and DAG. what happens next?

Answer 7

• exportin5 bound to RanGTP bind to pre-miRNA and translocate it into the cytoplasm via nuclear pore
• once on other side, RanGAP causes RanGTP -> RanGDP
• dissociation from pre-miRNA and exportin5 and RanGDP recycled back to the nucleus
• DICER (free in cytoplasm) can bind and process the pre-miRNA -> miRNA

Question 8

name the DICER substrates and draw a diagram showing this DICER reaction

Answer 8

• longdsRNA
• endogenous miRNA
• all products around 25nts long

Question 9

give the full name of the AGO protein

Answer 9

argonaute protein

Question 10

what are the 2 pathways that can create substrates which can be bound by the AGO protein

Answer 10

• RNAi pathway (from long dsRNA)

- microRNA pathway (from pre-miRNA)

Question 11

what is the function of the AGO protein? how does it achieve this?

Answer 11

• the AGO protein unwinds the DNA duplex by the least stable end. most unstable end because higher proportion of UG/UA bps (low GC content - this is the stronger bp)
• the AGO protein incorporates the strand whose 5’ end least stably pairs with the passenger strand
• once AGO bound to the single strand (guide strand complementary to the mRNA target) the RISC complex is formed

Question 12

draw a diagram showing the possible mechanisms of post-transcriptional gene repression by the microRNP complex

Answer 12

325 - 4 word

Question 13

what is the principal mode of action of siRNAs?

Answer 13

trigger degradation of the target miRNA

Question 14

where are the majority of miRNA binding sites?

Answer 14

majority within the 3’ UTR

Question 15

draw a diagram showing the non specific silencing upon viral infection in mammalian cells

Answer 15

325 - 4 word

Question 16

why is it important when talking about RNA interference to now say a gene is KNOCKED OUT, what should we say instead?

Answer 16

from siRNA, only about a 90% protein reduction, never 100

- always still some protein expressed therefore we say the gene has been KNOCKED DOWN

Question 17

instead of injecting in long dsRNA as siRNA, what is an alternative to this?

Answer 17

• plasmids encoding miRNA mimics
• contain inducible promoters therefore we can get inducible RNA interference
• therefore we can create stable RNAi cell lines (as the plasmid enters the progeny) for the essential genes and induce the RNAi response when we want

Question 18

describe and DAG explaining how adult C. elegant worms can take up RNAi

Answer 18

• E. coli contains plasmid with T7 promoters firing in both directions. convergent transcription allows us to achieve dsRNA against specific C elegant gene
• C. elegans eggs added to plate that E. coli living on and C elegans eats this, E. coli reaches gut, lysed and releases dsRNA
• analyse adult worms for F1 phenotype

Question 19

how was the process of a dsRNA library generated by bacteria?

Answer 19

• bacteria expressing dsRNA
• around 19,000 primer pairs that each code for a protein coding gene obtained PCR products and cloned into dual promoter vector
• both sense and antisense transcribed -> dsRNA
• reached 86% of genes (17,000 different strains)
• not 100% because of PCR failures, cloning failures

Question 20

what does the diagram in q20 325 -4 word show?

Answer 20

• RNAinterference against daf-2 gene knocks it down

- results in more obese worm as we see increased Nile Red staining (which reveals fat)