Lecture 19

Question 1

Dicer processes long dsRNA in the cytoplasm to produce:

Answer 1

• multiple products around 21-25nt long
• 2nt 3’ overhands
• 5’ phosphates

Question 2

Viral sources of dsRNA

Answer 2

• Most plant and animal viruses have:
• RNA virus, encoded by RNA-dependent RNApol, dicer chops it into primary viral siRNAs that are loaded into siRISC
• DNA viruses use bidirectional transcription to produce overlapping transcripts, chopped by dicer to produce primary viral siRNAs that are loaded into siRISC

Question 3

Viral defence in flies:

Answer 3

• siRNAs are produce when cells are infected with virus
• Drosophila s2 cells are infected with flock house virus (FHV)
• Around day 2 and day 3 maximum production of siRNAs is measured leading to a decrease in transcripts
• Dicer mutants render cell lines hypersensitive to FHV

Question 4

RNA interference in viral infection in plants - recovery:

Answer 4

• plants succumbing to virus infection will often produce leaves free of symptoms
• This is because the new leaves are resistance to a 2nd infection from the same virus.
• This is RNA interference, due to siRNAs targeting the viral genome

Question 5

What does systemic spread of RNAi imply?

Answer 5

• That siRNA amplification is occuring
• This occurs when SID1, a membrane transporter, moves RNA from cell to cell in worms.
• In plants the movement of RNA interference moves long distance through veins, and cell to cell via pores

Question 6

RNA-dependent RNA polymerases:

Answer 6

• Amplification of siRNAs requires RNA-dependent RNA polymerase (RDR)
• secondary siRNAs arise by two mechanisms - dicer dependent (in plants) and dicer independent (in worms)

Question 7

Viral immunity in mammals:

Answer 7

• Non-specific response to viral dsRNA and exogenous dsRNA
• toll like surface receptors recognise dsRNA/DNA motifs
• Type 1 interferons and RNases are produced
• This is innate immunity

Question 8

Schizosaccharomyces pombe (fission yeast)

Answer 8

• Dicer dcr1
• Argonute - argo1
• RDR - rdp1
• All the ingredients for RNA interference
• mutations in these components grow poorly due to expression of centromeric repeats, reduction in histone H3 lysine 9 methylation at the centromere and are defective in chromosome segregation

Question 9

RITS complex binds to:

Answer 9

• RNAPIII
• Forms an assembly platform on nascent transcript
• recruits RDR/dicer - generating secondary siRNAs
• This recruits histone methyltransferase, k9 of H3 tails are methylated
• Swi6 is involved in heterochromatin formation and spread

Question 10

Features of chromatin RNAi, it involves transcriptional gene silencing:

Answer 10

• nuclear not cytoplasmic
• siRNA’s are generated form dsRNA (endogenous source)
• siRNAs are loaded into AGOs that are specific for chromatin RNAi
• AGO-containing complex recruit chromatin/DNA modifying enzymes
• Gene silencing is achieved by repressing transcription through chromatin modification
• Targets transposons and other repetitive elements

Question 11

Do all transposons produce dsRNA?

Answer 11

NO!

Question 12

Applications of RNAi:

Answer 12

• Any gene in the genome can be targeted as all cells undergo and every cell can elicit RNAi
• Study of gene function (reverse genetics)
• Therapeutics (Target mRNA from disease causing genes/viruses)
• Biotechnology (metabolic engineering)

Question 13

RNAi induction involves two possible methods:

Answer 13

• Introducing chemical synthesized dsRNA into an organism

- Produce dsRNA molecules in vivo

Question 14

Artificially generated dsRNA:

Answer 14

• In vitro synthesis of dsRNA: long dsRNA is processed by dicer which is more effective
• Modification of dsRNA prevents degradation in vivo, by introducing a 2’ base sugar modification or a backbone modification

Question 15

Delivery of synthesized dsRNA after modifying it:

Answer 15

• Add cholesterol to 5’ end of sense srang
• Encase dsRNAs in lipid sphere
• Protamine-antibody (attach it to an antibody fragment, allowing targeting of particular cell surfaces.

Question 16

Synthesis of dsRNA in vivo (endogenously):

Answer 16

• Use of retrovirus vectors (genome is protected in the plasma membrane).
• Take vector and generate a short hairpin, so reverse sequence complementarity allows base pairing in the DNA
• Modified virus will fuse with the cell and the DNA will be released and hopefully integrate into the host genome
• substrate for dicer and produces siRNAs.

Question 17

We can use RNAi to study gene function:

Answer 17

• Advantage over conventional mutagenesis
• Reduce but not eliminate gene activity
• Not confined to model organisms
• Under for functional genomics (high throughput studies)

Question 18

RNAi can be used to treat various diseases, eg) age related macular degeneration

Answer 18

• The macular (visualising small detail) degeneration, proliferation of blood vessels causes them to start bursting more often.
• Increased expression of vascular endothelial growth factor (VEGF) causes proliferation
• Eliminate the activity of VEGF by lazering the eye, and introducing siRNA

Question 19

siRNAs:

Answer 19

• Derived from exogenous dsRNAs, repetitive elements and viruses
• Arise from long, perfectly complementary dsRNA
• Show extensive complementarity to their targets
• Slive target mRNA, cytoplasmic RNAi histone/DNA modification and chromatin RNAi

Question 20

miRNAs:

Answer 20

• Encoded by distinct genes within the genome
• Derived from imperfect step-loop precursors
• Show limited complementarity to their targets (animals)
• Block translation and cause mRNA decay (animals)