Lecture 21

Question 1

What does removing a protein do?

Answer 1

Removing a protein informs us of its function

Question 2

What does the eyeless protein do?

Answer 2

Eyeless protein coordinate the formation of eyes, without it we would not have eyes.

Question 3

What does the white protein do?

Answer 3

The white protein contributes to eye pigmentation, making the eye white.

Question 4

What is the central dogma of biology?

Answer 4

DNA -> RNA -> protein

Question 5

How does our genome operate?

Answer 5

Out genome operates by sending information from double stranded DNA in the nucleus, via single stranded mRNA, to guide the synthesis of proteins in the cytoplasm

Question 6

What is hybridization?

Answer 6

Hybridization is the process of combining two complementary single-stranded DNA or RNA molecules and allowing them to forma single double-stranded molecule through base pairing.

Question 7

What does RNA hybridization allow?

Answer 7

RNA hybridization allows the detection of mRNA molecules

Question 8

What method do we use to detect mRNA?

Answer 8

1. Target hybridization: addition of an oligonucleotide (basically DNA sequence) that is attached to a fluorophore is added to a strand of RNA
2. Fluorescence imaging: allows us to actually see the mRNA molecules in a cell.

Question 9

WHat is the effect of adding sense RNA to a parent?

Answer 9

The result of adding sense RNA to a parent is of no effect in the offspring.
- Sense RNA is the typical RNA we use to synthesize proteins

Question 10

What is the result of adding antisense RNA to a parent?

Answer 10

The result of adding antisense RNA is that the RNA would be affecting mRNA, but only when able to affect the specific proteins.
- In our example, there is no difference on the offspring.

Question 11

What is the result of adding double stranded RNA to a parent?

Answer 11

The result of adding double stranded RNA to a parent results in a twitching offspring. The ribosome is not able to attach to the dsRNA and therefore cannot make the protein, causing damage to the cell.

Question 12

Effect of dsRNA vs. ssRNA?

Answer 12

RNa carrying the code for a muscle protein is injected onto the worm. Single stranded RNA has no effect, but double stranded RNA injection leads to the worm twitching in a s similar way to works carrying a defective one for the muscle protein.

Question 13

What is the effect of dsRNA on protien synthesis?

Answer 13

The injection of dsRNA inhibits synthesis of specific proteins.
- If you inject dsRNA, cells will stop working for the functional purposes of those proteins.

Question 14

What does dsRNA promote?

Answer 14

The injection of dsRNA promotes mRNA degradation
- injection of antisense = less mRNA
- injection of dsRNa = no mRNA

Question 15

What is RNAi?

Answer 15

RNA interference (RNAi) is a mechanism of gene regulation

Question 16

What is the mechanism by which dsRNA degrades mRNA?

Answer 16

1. Double stranded RNA (dsRNA) binds to the protein dicer
2. dicer cleaves dsRNA into smaller fragments
3. One of the fragments/RNA strands os loaded into a RISC complex
4. RISC complex links the mRNA and the fragment by base pairing
5. mRNA is cleaved and destroyed and no protein is synthesized.

Question 17

Is dsRNA normal?

Answer 17

No, dsRNA is not something that we normally have.

Question 18

What is dicer and what is its role?

Answer 18

Dicer is a nuclease enzyme that cleaves the dsRNA into small fragments of approximately 23 nucleotide pairs that are called small interfering RNAs (siRNAs)

Question 19

How else can we get siRNA in our cells?

Answer 19

We can also just inject siRNAs for specific genes if we want.

Question 20

siRNAs are bound by what?

Answer 20

The double-stranded siRNAs are then bound by Argonaute and other components of the RISC complex.

Question 21

What is the RISC complex?

Answer 21

The RNA-induced silencing complex (RISC) is a complex of ribonucleic
acid and RNA-binding proteins.

Question 22

What is the RISC complex composed of?

Answer 22

The RISC complex is composed of Argonaute, RNA and RNA-binding proteins.

Question 23

Summary of mRNA degradation by dsRNA?

Answer 23

1. Long double-stranded RNA (dsRNA) is cleaved into small interfering RNA (siRNA) by the enzyme Dicer.
2. The siRNA joins the RNA-induced silencing complex (RISC),
3. Argonaute 2 (AGO2) cleaves the sense strand of RNA .
4. The RISC–siRNA complex binds and degrades complementary mRNA
   ●5. The activated RISC–siRNA complex can then be reused

Question 24

Why might we introduce dsRNA experimentally?

Answer 24

Double-stranded RNA can be introduced experimentally to silence genes of interest
- We can essentially remove any protein experimentally using dsRNA.

Question 25

what molecule mimics dsRNA?

Answer 25

A short hairpin RNA shRNA is an artificial RNA molecule with a hairpin turn that mimics a dsRNA molecule

Question 26

RNAi allows us to remove proteins without....?

Answer 26

RNAi allows remove specific proteins in cells and tissues without affecting the genome

Question 27

How is RNAi used therapeutically?

Answer 27

Therapeutic ○ Remove malfunctioning proteins that cause problems ○ Destroy viruses

Question 28

How is RNAi used for research?

Answer 28

Research ○ Study the functions of individual proteins

Question 29

How did they discover pressure-gated channels?

Answer 29

Pressure-gated channels were discovered through gene silencing by using RNAi and inhibiting the production of certain proteins. - Channels like PIEZO were discovered this way

Question 30

How is RNAi involved in curing Huntington's disease?

Answer 30

Huntington’s is caused by mutations in huntingtin gene ● The mutated huntingtin proteins accumulate in neurons and cause brain damage * Therapeutic silenting of this mutant with siRNA has been able to attenuate many affects of this disease and effecitvely make it less harmful for people with it.

Question 31

How do we identify cell morphology proteins?

Answer 31

An RNAi screen in cells to identify cell morphology proteins. There are four steps as follows: 1. Identify an interesting process → e.g., cell morphology 2. Get a library of dsRNA that cover all the genes 3. incubate cells with specific dsRNA 4. score the changes