RNA Interfer ence (RNAi)

1. RNAInterference(RNAi) By Shamii Goh By Shamii Goh

2. RNAi The Purpose: • Silences gene expression post-transcription • Found naturally in cells to ward off "aberrant RNA" or dsRNA and degrade this RNA to protect themselves from transposition and virus infection through the use of the RNA interference mechanism

3. The Science: • Step 1: A double strand of RNA (dsRNA), normally rare in cell, is introduced (either by a researcher, virus, or transposon) • Step 2: An enzyme called “Dicer” “cleaves” the RNA into pieces around 21-25 base pair long molecules which are called “small interfering RNA” (siRNA) and are also known as “guide RNA” (at this point they may or may not be double or single stranded and they may be amplified).

4. Step 3: The siRNA bind to some proteins (the main one is Argonaute) to form an RNA-induced silencing complex (RISC). • Step 4: The siRNA direct RISC to the target homologous cellular mRNA which binds to the gene through complementary base pairing. Then the other proteins degrade the rest of the mRNA strand so that it cannot produce more proteins using exonucleolytic or endonucleolytic cleavage. This process is basically “sequence specific gene silencing”.

5. Step 5: It is proposed that the SiRNAs are also used as primers for the next generation of dsRNA for RNAi possibly made by the enzyme RNA polymerase (RdRp).

6. Lab Technique: • First, dsRNA with the unwanted gene is synthesized in the lab using RNA polymerase and PCR • The dsRNA gets injected into the targeted cell by a variety of ways including microinjection, electroporation, or even, with C.elegans, by soaking the organism in dsRNA solution. It can also be introduced into the worm when it ingests bacteria that express dsRNA from recombinant plasmids. • Then the scientific part happens (see slides 3-5) • And the organism either does not express the gene that has been inhibited or it does.

7. Applications: • Naturally, RNAi is important for protection of the genome from viruses and “jumping genes” and for regulating gene expression (for example, it guides embryo development by turning down specific genes at critical times)

8. Better Applications • Revolutionary! • Used in “reverse genetics” •  When developed further can be used to stop just about any inherited or gene related disorder/illness including HIV, some types of cancer, arthritis, and macular degeneration of the eye because we can insert a double strand of the gene that causes the disease and then the actual gene will degenerate RNAi has been shown to work in a study of stopping the development of hepatitis in living mice

9. More Better Applications… • Can also be used for figuring out what specific genes do : i.e. removing gene # 1 and seeing the effect it creates in an organism- • for example, a petunia with no color.

10. A Few Weird Words: the Glossary • Exonucleolytic: cleaving a nucleotide chain at a point adjacent to one of its ends • Endonucleolytic: cleaving a nucleotide chain into two parts at an internal point • Transposon/“jumping gene”: a sequence of DNA that can move around to different positions within the genome of a single cell, a process called transposition. In the process, they can cause mutations and change the amount of DNA in the genome.

11. The Nobel Prize 2006 Andrew Fire Craig Mello