RNAi and siRNA

Question 1

What was discovered by Fire and Mello experiment?

Answer 1

double-stranded RNA was substantially more effective at producing interference than was either strand individually

Question 2

What are important characteristics of RNAi in C.elegans?

Answer 2

• RNA must be double stranded
• introns and promoters do not work, must be from the mRNA but not limited to 3’UTR
• decrease in endogenous mRNA
• a small amount of injected and a large number of cells displaying phenotype change
• heritable
• may have cross-effects if a multi-gene family
• dsRNA can cross cellular boundaries. Suggests RNA-transport mechanism

Question 3

What are the steps of dsRNA production?

Answer 3

1. Segments are cloned between flanking copies of the bacteriophage t7 promoter into a bacterial plasmid vector
2. a bacterial strain expressing the t7 polymerase gene from an inducible promoter was used as a host. Alternatively a single copy of the t7 promoter to drive expression of an inverted duplication for a segment of the target gene

Question 4

What are the ways to introduce RNAi?

Answer 4

• injection
• internal expression
• soaking
• feeding

Question 5

What is the difference between miRNA and the siRNA pathway?

Answer 5

the unequal degree of complementarity between the small RNA and the target mRNA

Question 6

What is the RNAi mechanism steps?

Answer 6

1. double stranded RNA binds to the protein dicer
2. the dicer cleaves dsRNA into smaller fragments
3. one of the RNA strands is loaded into a RISC complex
4. the RNA strand links the complex to the mRNA strand by base pairing
5. mRNA is cleaved and destroyed. No protein can be synthesized

Question 7

What are some uses of the RNAi mechanism?

Answer 7

• when an RNA virus infects the cell, it injects its genome consisting of double-stranded RNA. RNA interference destroys the viral RNA, preventing the formation of new viruses
• synthesis of many proteins is controlled by genes encoding microRNA. After processing, micrRNA prevents the translation of mRNA to protein
• in the research laboratory, dsRNA molecules are tailor-made to activate the RISC complex to degrade mRNA for a specific gene