Lecture 28 - RNAi Knockdown

Question 1

How do we know about the function of most proteins? Give the 2 examples given

Answer 1

• by removing one protein at a time and seeing what it affects
• eyeless protein coordinates the formation of eyes (flies)
• white protein contributes to eye pigment (flies)

Question 2

Central Dogma

Answer 2

DNA -> mRNA -> protein

our genome operates by sending info from dsDNA in nucleus, via ssmRNA, to guide synthesis of proteins in the cytoplasm

Question 3

Hybridization

Answer 3

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

Question 4

What does RNA hybridization allow?

Answer 4

• the detection of mRNA molecules
  1. target hybridization: an oligonucleotide finds and attaches to mRNA => a fluorophore conjugates to the oligonucleotide
  2. detect fluorescence using fluorescence imaging

Question 5

What is RNA interference?

Answer 5

RNA interference (RNAi) is a natural process where cells can “turn off” specific genes. It uses small RNA molecules that interfere with certain mRNAs being turned into proteins, effectively silencing those genes.

Question 6

What was the example used to show that dsRNA has a strong effect?

Answer 6

• sense RNA had no effect on the offspring of C elegans
• antisense RNA also had no effect
• dsRNA (i.e., sense and antisense together) caused twitching in the offspring

Question 7

What happens when you inject dsRNA?

Answer 7

• it inhibits the synthesis of specific proteins by promoting degradation of mRNA that would encode that protein

Question 8

RNA interference (RNAi) is a mechanism of ______ __________

Answer 8

gene regulation

Question 9

Briefly explain what happens after dsRNA is injected

Answer 9

1. dsRNA binds to the protein Dicer, which cleaves dsRNA into smaller fragments
2. one of the RNA strands is loaded into the RISC complex
3. the RNA strand links the complex to an mRNA strand by complementary base pairing
4. mRNA is cleaved and no protein is synthesized

Question 10

Dicer

Answer 10

• a nuclease enzyme (cuts nucleic acids)
• cleaves dsRNA into small fragments (approximately 23 nucleotide pairs) called small interfering RNAs (siRNAs)

Question 11

What happens the small interfering RNAs after being cleaved by Dicer?

Answer 11

they are then bound by Argonaute and other components of RISC complex

Question 12

What ion associates to the Dicer protein?

Answer 12

manganese

Question 13

What is the RISC complex? How does it work?

Answer 13

• RNA-induced silencing complex (RISC)
• composed of Argonaute, RNA and RNA-binding proteins
• one of the RNA strands (siRNA) is loaded into the complex and links it to the mRNA strand by base pairing

Question 14

What are the 3 processes in the cell that use RNAi that we talked about?

Answer 14

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

Question 15

What is a short hairpin RNA (shRNA)?

Answer 15

• an artificial RNA molecule with a hairpin turn that mimics a dsRNA molecule

Question 16

Does RNAi allow removal of specific proteins in cells and tissues without affecting the genome? If so, give 2 examples of where/how it can be used.

Answer 16

yes

1. therapeutic: remove malfunctioning proteins that cause problems; destroy viruses
2. research: study functions of individual proteins

Question 17

Huntington’s Disease and RNAi

Answer 17

• huntington’s is caused by mutations in huntingtin gene
• mutated huntingtin proteins accumulate in neurons and cause brain damage
• therapeutic silencing of mutant huntingtin with siRNA can alleviate symptoms