L28 siRNAs RNAi Flashcards
siRNAs
defence against foreign genetic material e.g. viruses and transposons
small RNAs are associated with viral infections
small RNAs complementary to viral RNA detected in tissue of infected plants and animals
viral sources of dsRNA
see onenote
viral siRNAs created via dicer - primary siRNAs
viral defence improved with two processes involving RNA-dependent RNA polymerase (RDR)
see onenote
- amplification
- transitivity
RDR is host encoded
Amplification
see onenote
massive increase in siRNAs (secondary siRNAs)
occurs via positive feedback
Transitivity
see onenote
spreading of targeting beyond initial trigger region
Secondary siRNA, targeting other regions of viral RNA
Spreading out from initial targeting site
Recovery - evidence for systemic viral defence
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
response to viral infection
viral RNA cleaved by dicer and risc (slicer) activity
success = rate of slicing/dicing > viral replication
Primary siRNAs
derived from dicer activity
Systemy
see onenote
spread of siRNAs into other tissue
siRNA in worm/plants spreads from source of induction - systemic
systemic spread implies siRNA amplification
Viral immunity in vertebrates
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
transposons and genome integrity
trasposons threat to genome integrity
mechanisms evolved to suppress transposon activity
RNAi and defence against transposon
see onenote
screen for RNAi defects - bag of worm phenotype, pos-1 ds RNA not repressed
link between RNAi and transposon mobilisation
Tc1 transposon
see onenote
belongs to the mariner family of TEs
dsRNA derived from TIR and internal sequences of Tc1 - endogenous siRNAs
MuDR
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
RNAi and transcriptional gene silencing
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
Mechanism of chromatin RNAi
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
RITS complex forms an assembly platform
- recruits histone methyltransferases, chromatin remodelling factors (heterochromatin formation)
=> spread of heterochromatin
RNAi has multiple applications
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
Issues associated with RNAi induction
see onenote
in vitro synthesized dsRNA - introduction of dsRNA into organism
in vivo synthesized dsRNA - endogenous production of dsRNA in organism
Delivery of synthesized dsRNA
see onenote
dsRNA must traverse the cell membrane if it is to enter RISC
- add cholesterol
- encase dsRNA in lipid sphere
- protamine-antibody fusion, protamine made up of highly charged aa
Viral delivery
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
Therapeutic applications
see onenote
AMD example - age related macular degeneration
VEGF
- Target for RNAi
- Eliminate activity of VEGF => reduce incidence of AMD
