L28 siRNAs RNAi

Question 1

siRNAs

Answer 1

defence against foreign genetic material e.g. viruses and transposons

Question 2

small RNAs are associated with viral infections

Answer 2

small RNAs complementary to viral RNA detected in tissue of infected plants and animals

Question 3

viral sources of dsRNA

Answer 3

see onenote

viral siRNAs created via dicer - primary siRNAs

Question 4

viral defence improved with two processes involving RNA-dependent RNA polymerase (RDR)

Answer 4

see onenote

1. amplification
2. transitivity

RDR is host encoded

Question 5

Amplification

Answer 5

see onenote

massive increase in siRNAs (secondary siRNAs)

occurs via positive feedback

Question 6

Transitivity

Answer 6

see onenote

spreading of targeting beyond initial trigger region

Secondary siRNA, targeting other regions of viral RNA
Spreading out from initial targeting site

Question 7

Recovery - evidence for systemic viral defence

Answer 7

see onenote

plants succumbing to virus infection will often produce leaves free of symptoms

leaves resistant to second infection from same/closely related virus

Virus is spreading but so is siRNA
siRNA has spread faster than virus, protecting the leaves at the top
Plants have an immune system, RNAi pathway is an important path of that

Question 8

response to viral infection

Answer 8

viral RNA cleaved by dicer and risc (slicer) activity

success = rate of slicing/dicing > viral replication

Question 9

Primary siRNAs

Answer 9

derived from dicer activity

Question 10

Systemy

Answer 10

see onenote

spread of siRNAs into other tissue

siRNA in worm/plants spreads from source of induction - systemic

systemic spread implies siRNA amplification

Question 11

Viral immunity in vertebrates

Answer 11

see onenote

• non-specific response to viral dsRNA and exogenous dsRNA
• distinct sequence motifs recognised by membrane receptors and other cellular proteins
• binding triggers production of type 1 interferons and RNAses
• inferons affect viral replication

Question 12

transposons and genome integrity

Answer 12

trasposons threat to genome integrity

mechanisms evolved to suppress transposon activity

Question 13

RNAi and defence against transposon

Answer 13

see onenote

screen for RNAi defects - bag of worm phenotype, pos-1 ds RNA not repressed

link between RNAi and transposon mobilisation

Question 14

Tc1 transposon

Answer 14

see onenote

belongs to the mariner family of TEs

dsRNA derived from TIR and internal sequences of Tc1 - endogenous siRNAs

Question 15

MuDR

Answer 15

see onenote slides

mutator - DNA transposon of maize, most mutagenic eukaryotic transposon family known

activity of MuDr dominantly suppressed by Mu killer (MuK), associated with DNA methylation

MuK - inverted duplication of a partially deleted MuDR element => dsRNA hairpin MuK transcript can silence MuDR

TE inactivation involves transcriptional gene silencing

Question 16

RNAi and transcriptional gene silencing

Answer 16

see onenote

Gene silencing in fission yeast resulted in:

• 1 copy of dicer, 1 copy of AGO and 1 copy of RDR
• Mutate any one of these genes => affecting chromosome segregation, centromeric repeats become transcriptionally active, reduced H3K9 marks methylation at centromeres
• Mutants that can’t generate heterochromatin, RNAi in this organism somehow linked to heterochromatin formation
• Heterochromatin formation may be linked to formation of endogenous siRNA

Question 17

Mechanism of chromatin RNAi

Answer 17

see onenote slides

Centromeric repeat

• Convergent transcription from both strands => long perfectly complementary DS RNA => dicer => siRNA
• RITS enters nucleus, affects activity of centromeric repeat
• RITS bind to nascent transcript, cause slicing of that transcript => becomes substrate of RDR
• Binding of RISC to nascent transcript forms assembly platform

Question 18

RITS complex forms an assembly platform

Answer 18

• recruits histone methyltransferases, chromatin remodelling factors (heterochromatin formation)
  => spread of heterochromatin

Question 19

RNAi has multiple applications

Answer 19

see onenote

RNAi can target any gene as long as siRNA or their dsRNA precursors can be introduced into the cell

study of gene function
therapeutics
biotechnology

Question 20

Issues associated with RNAi induction

Answer 20

see onenote

in vitro synthesized dsRNA - introduction of dsRNA into organism

in vivo synthesized dsRNA - endogenous production of dsRNA in organism

Question 21

Delivery of synthesized dsRNA

Answer 21

see onenote

dsRNA must traverse the cell membrane if it is to enter RISC

1. add cholesterol
2. encase dsRNA in lipid sphere
3. protamine-antibody fusion, protamine made up of highly charged aa

Question 22

Viral delivery

Answer 22

see onenote

viral vectors generate short hairpin RNAs (shRNAs) when expressed in vivo - short/long term RNAi

Viral vector
- Viral genome surrounded by viral capsid, not exposed to nucleases, won’t illicit non-specific immune response that otherwise would be triggered due to exposure of dsRNA

Question 23

Therapeutic applications

Answer 23

see onenote

AMD example - age related macular degeneration

VEGF

• Target for RNAi
• Eliminate activity of VEGF => reduce incidence of AMD