W8L1 Function of siRNAs and their application

Question 1

Small RNAs and viral infection

Answer 1

• small RNAs complementary to viral RNAs are detected in tissue of infected plants and animal

Question 2

Viral sources of dsRNA

Answer 2

RNA virus, DNA virus and structured viral genome or mRNA can be processed into viral siRNAs by dicer
-RNA virus can make replication intermediate
-DNA virus have bidirectional transcription, overlapping transcription double stranded

Question 3

siRNA Respond to viral infection

Answer 3

Viral RNA is targeted by a combination of Dicer and RISC (slicer) activity
-load into Argo, viral RISC which cleave ss viral RNA
- the rate of slicing must be higher than viral replication

Question 4

Amplification and transitivity

Answer 4

Viral defence enhanced by TWO processes involving RNA-dependent RNA polymerase (RDR):
§ Amplification – massive increase in siRNA production (2o siRNAs). These 2o siRNA bind to ss viral RNA, creating a double-stranded, target for dicer

§ Transitivity – spreading of siRNAs beyond the initial trigger region

Question 5

Recovery – adaptive immunity in plants

Answer 5

Plants succumbing to virus infection will often produce NEW leaves free of disease symptomsNewly emerging leaves are resistant to a 2nd infection by the same or closely related virus

Question 6

RNAi is systemic

Answer 6

RNAi in worms/plants spreads from source of induction
– systemicGFP expressing worms fed dsRNA GFP, spreads from cell to cell via membrane transporter SID1
-In plant spread from cell to cell via pores and vascular network in long distanceSystemic RNAi implies siRNA amplification

Question 7

RNAi is not part of vertebrate viral immunity

Answer 7

§ Non-specific response to viral dsRNA and exogenous dsRNA
§ Distinct sequence motifs recognized by membrane receptors and other cellular proteins
§ Binding triggers production of type I interferons and RNases
§ Interferons disrupt viral replication

Question 8

Transposable elements (TE) and genome integrity

Answer 8

TE present a potent threat to genome integrity:
Mutagenic potential
Chromosome breakage
Ectopic recombination
Replicative load
Mechanisms have evolved to suppress TE activity

Question 9

Mobility of TE is suppressed in the germline of the worm

Answer 9

In nematodes there are 32 copies of Tc1, a DNA transposon belonging to the mariner family of class II TEs -Tc1 have a 54bp with terminal inverted repeat-unc-22/Tc1 mutant lead to twitcher phenotype-observe low level excision of Tc1 in somatic tissue but all progeny have the twitcher phenotype-There is a germ line specific mechanism for TE silencing

Question 10

Mutant screen for genes involves in TE silencing

Answer 10

Screen to identify mutants that are no longer able to silence Tc1 in the germline
Use unc-22/Tc1 mutant background-most offspring have twitching phenotype but some have WT movement arise from transposition
Mutant screens identified over 25 genes that enable Tc1 to transpose in the germline
Many of these genes are components of the RNAi pathway (dicer/RISC)

Question 11

how can TEs be a potent source of dsRNA

Answer 11

-TE adjacent to an endogenous promoter, leading to a long dsRNA, process by Dicer
-Readthrough from an adjacent endogenous promoter lead to short stem loop
-readthrough into an adjacent TE, short stem loop
-insertion of inverted TE lead to long stem loop

Question 12

RNAi and defence against transposons in the germline of worms

Answer 12

A few Tc1 elements are adjacent to genes in the genomes dsRNA generated to these Tc1 elements
– endo-siRNAs produced by DicerAmplification/transitivity
– large pool of endo-siRNAs to Tc1 elements-targeting of transposable mRNA limits Tc1 excision in the germ line

Question 13

Evidence of RNAi is associated with transcriptional regulation

Answer 13

mutation in Single Dicer –dcr11defects in chromosome segregation
mutation in ago1 Single Argonaute –expression of centromeric repeats
mutation in rdp1 1Single RDR – reduced histone H3K9 methylation marks at centromeresSuggests that:
RNAi is associated with heterochromatin formation
Source of endo-siRNA from centromeres
RNAi working as an epigenetic modifer

Question 14

Mechanism of heterochromatin formation due to RNAi

Answer 14

-Centromeric repeats transcribed from both strands by RNA pol II
-Nascent transcripts anneal to form long dsRNA
-Processed into endo-siRNAs in the cytoplasm
-siRNAs loaded into an RNA-induced transcriptional silencing (RITS) complex-Complex enter the nucleus and binds to a nascent transcript and slices-Recruits RDR
– generates 2o siRNAs -RITS from an assembly platform, histone methyltransferases recruited
-H3K9 methylation-Chromatin remodeling factor recruited to H3K9 tails-Nucleation site for heterochromatin formation spread for several kbs

Question 15

Applications of RNAi

Answer 15

RNAi can target ANY gene provided siRNAs or their dsRNA precursors can enter the cel
l-useful to study of gene function, therapeutics and biotechnology

Question 16

induction of RNAi: in vitro

Answer 16

In vitro synthesized dsRNA – introduction of dsRNA into the organism
§ Stability – protect against ribonucleases and host immune response
§ Delivery into the cell – cross cell membrane
§ Transient RNAi in organisms without siRNA amplification

Question 17

induction of RNAi: in vivo

Answer 17

In vivo synthesized dsRNA – endogenous production of dsRNA in organism
§ dsRNA producing construct within the cells of an organism
§ Highly stable and no issues with crossing cell membrane
§ Long term RNAi

Question 18

Delivery of exogenous siRNAs

Answer 18

1. dsRNA must be protected from ribonucleases in the circulatory system
2. dsRNA must traverse the cell membrane if it is to enter RISC
   -add cholesterol in the 5’end of the sense strand
   -encase dsRNA in a carrier protein
   -antibody fusion

Question 19

Generating endo-siRNA from shRNAs

Answer 19

Recombinant retroviral vectors generate short hairpin RNAs (shRNAs) when expressed in vivo
–short/long term RNAi
– adenoviruses Packaged recombinant retrovirus
Genetic cargo protected from ribonucleases

Question 20

Mouse model for AMD

Answer 20

• Induce sub-retinal vascularization (neovascularization) in mouse eye using laser treatment is associated with increased expression of VEGF* Treatment with naked double-stranded VEGF siRNA effectively blocks neovascularization

Question 21

Problems with an RNAi approach

Answer 21

1. ds-siRNAs have a non-specific effect on neovascularisation due to the triggering of an innate immune reaction
2. Introduction of siRNA into cells causes upregulation of miRNA-controlled genes