Lecture 20: RNA Interference

Eye Pigmentation

• eyeless protein coordinates the formation of eyes

• white protein contributes to eye pigmentation

RNAi

• post-transcriptional gene-silencing mechanism where double-stranded RNA (dsRNA) leads to the degradation of specific mRNA molecules.

  • Result: protein levels go down without changing the genome

• mechanism of gene regulation

  • allows the removal of specific proteins in cells/tissues without affecting the gene

Hybridization

• process of combining 2 complementary single-stranded DNA or RNA molecules and allowing them to form a single, double stranded molecule through base pairing

• allows for the detection of mRNA molecules via:

  • target hybridization

  • fluorescence imaging

RNA Injection Experiments

• injection of double stranded RNA has a strong effect

  • dsRNA → loss of target protein

  • dsRNA → promotes mRNA degradation

• No staining / no protein detected

• Specificity: only the targeted gene is affected

siRNAs

• dicer cleaves dsRNA into small interfereing RNAs (siRNAs)

• then, siRNAs are bound by Argonaute and other components of the RISC complex

RISC Complex

• RNA induced silencing complex

• complex of ribonucleic acid and RNA binding proteins

RNAi Pathway

1. Double-stranded RNA (dsRNA) enters the cell

2. Dicer (an RNase enzyme) cleaves dsRNA into:

   • siRNAs (~23 nucleotide pairs)

3. siRNA binds to Argonaute (AGO) proteins

4. siRNA + Argonaute + other proteins form the RISC complex

5. Argonaute 2 (AGO2):

   • Cleaves the sense strand

   • Leaves the antisense (guide) strand

6. The guide strand base-pairs with complementary mRNA

7. RISC degrades the target mRNA

8. RISC is reused to silence more mRNA molecules

RNAi Applications

• Theraputic

  • remove malfunctioning proteins that cause problems

  • destroy viruses

• research

  • study the functions of individual proteins

• Huntington’s disease potential cure

shRNA

• short hairpin RNA

• artifical RNA molecule with a hairpin turn that mimics a dsRNA molecule

• Used for:

  • Stable knockdown

  • Long-term experiments

RNAi Screening

Steps:

1. Identify a biological process (e.g., cell morphology)

2. Use a library of dsRNA/shRNA targeting many genes

3. Treat cells with individual RNAs

4. Score phenotypes (shape, movement, survival)