13. RNAi and viruses Flashcards
What is RNAi an essential form of viral defence in?
- Vertebrates
- Plants
- Insects
What types of RNAs can induce an RNAi response?
- dsRNA
- Short hairpin RNA
- Imported siRNA
What is siRNA?
- Short interfering RNA
- It is derived from an infecting virus
- It inhibits expression of 1 specific virus.
- Cleaves miRNA
What is miRNA?
- Micro RNA
- Produced from the host cell genome
- Regulates gene expression of multiple mRNAs
- Inhibits translation.
What are the key steps in the RNAi response?
- Dicer detects and cleaves up long dsDNA to produce siRNAs.
- siRNAs are then loaded into the RNA induced silencing complex (RISC).
- This leads to cleavage or degradation of viral RNA molecules and suppression of translation.
What is dicer?
- A key molecule involved in small RNA biogenesis.
- It is an RNA endonuclease (RNase).
- It can cleave siRNA and miRNA.
How is miRNA generated?
- miRNA is produced in all mammalian cells to regulate gene expression.
- It is produced as a hairpin and first cleaved by Drosha in the nucleus which produces 3’ overhangs.
- the miRNA is then exported to the cytoplasm and cleaved by dicer.
- This dicer cleavage generates the characteristic 22 nucleotide long miRNA.
- miRNA also have 2 nt 3’ overhangs which can bind to PAZ domains.
How is siRNA generated?
- Viral RNA like dsRNA or short hairpin RNA is introduced to the cell.
- Dicer cleaves the long RNAs to produce siRNA.
- These can then be loaded into the RISC complex.
What domains do dicer and drosha contain?
- Helicase binding domain to recruit helicases and unwind RNA.
- RNA binding domain to bind to dsRNA.
- PAZ domain which recognises and binds 3’ overhangs on siRNAs.
How does Dicer cleave dsRNA?
- It binds to to dsRNA through its binding domain.
- It recognises specific sequences for cleavage.
- This leads to specific RNA fragments generated due to the shape of the enzyme and interactions.
- These RNA fragments are always 22 nucleotides long.
How can tell if there is dicer activity in a cell?
- If you isolate and sequence all the RNA in the cell, you can tell the length of the fragments.
- If there is a massive abundance of 22nt long RNA fragments, you know there is dicer activity.
How does the RISC complex assemble using siRNAs?
- Dicer and argonaut 2 are the key molecules in this pathway.
- siRNAs are generated from viral RNA by dicer.
- R2D2 binds to the 5’ end of the siRNA and stabilise the molecule.
- Other enzymes also can modify the RNA at this stage
- Ago-2 recruits the siRNA to the RISC complex.
- Ago-2 also unwinds the RNA into the separate passenger and guide RNA strands.
- The passenger strand is destroyed.
- The RISC complex is then fully activated wih a guide RNA which can bind foreign RNA that is invading the cell.
What is the function of RNAi, siRNA and RISC complex?
To identify and destroy foreign RNA to prevent infection.
What is the structure of Ago-2?
- PIWI domain is an endonuclease that cleaves RNA.
- PAZ domain to bind to the 3’ end of the guide RNA
- These work together to recognise and bind viral RNA then cleave it.
How can siRNAs be used in experimental systems?
- They can be used to silence gene expression of specific genes.
- We can introduce siRNAs to destroy mRNAs to knock down gene expression.
How does the RISC complex cleave foreign RNA?
- RISC binds to ssRNA like mRNA through complementary base pairing between the guide RNA and target RNA.
- If the binding is totally complementary this results in cleavage and degradation.
- Non total complementary binding leads to translational suppression similar to miRNA system.
How can the RNAi response be amplified?
Plants and worms can generate RNA-dependent RNA polymerase that functions in the siRNA pathway.
How is the RNAi response amplified in plants?
- The primary cleavage by dicer produces siRNAs.
- RISC complex cleavage and formation then binding to target ssRNA.
- This target ssRNA is then made dsRNA by RNA-dependent RNA polymerase.
- this dsRNA is then used to generate a 2nd round of siRNAs and amplify the response.
What are the key points of the amplification of the RNAi response?
- Dicer cleaves the dsRNA to make siRNA.
- Formation of the RISC complex by Ago-2.
- A 2nd wave of siRNAs generated by RNA-dependent RNA polymerase.
How is the RNAi response amplified in worms?
- Worms don’t need the initial dicer cleavage to generates the RISC complex.
- RISC just binds to the target RNA
- The polymerase extends the RNA to make short dsRNA that then enters Ago-2.
What organisms is the RNAi response essential for?
- It is important in viral response in plants, arthropods and nematodes.
- dsRNAs are produced in viral infection which triggers RISC complex formation.
- This primes the organism to prevent viral infection.
Do mammals use the RNAi response?
- Mammals have all the components for siRNA recognition and defence.
- However they also have the more evolved type 1 interferon system that detects dsRNA and triggers a systemic interferon response through TLRs and RLR.
- When 1 cell is targeted it can protect the rest of the organism
Is RNAi a systemic response?
- Yes
- The siRNA response has been shown to prevent viruses spreading systemically around plants.
Can viruses suppress the RNAi response?
- Yes
- Even simple viruses can encode proteins that can suppress the RNAi response.