Lecture 19 Flashcards Preview

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Flashcards in Lecture 19 Deck (20):
1

Dicer processes long dsRNA in the cytoplasm to produce:

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

2

Viral sources of dsRNA

- 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

3

Viral defence in flies:

- 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

4

RNA interference in viral infection in plants - recovery:

- 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

5

What does systemic spread of RNAi imply?

- 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

6

RNA-dependent RNA polymerases:

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

7

Viral immunity in mammals:

- 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

8

Schizosaccharomyces pombe (fission yeast)

- 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

9

RITS complex binds to:

- 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

10

Features of chromatin RNAi, it involves transcriptional gene silencing:

- 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

11

Do all transposons produce dsRNA?

NO!

12

Applications of RNAi:

- 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)

13

RNAi induction involves two possible methods:

- Introducing chemical synthesized dsRNA into an organism
- Produce dsRNA molecules in vivo

14

Artificially generated dsRNA:

- 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

15

Delivery of synthesized dsRNA after modifying it:

- 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.

16

Synthesis of dsRNA in vivo (endogenously):

- 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.

17

We can use RNAi to study gene function:

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

18

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

- 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

19

siRNAs:

- 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

20

miRNAs:

- 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)