RNA interference

What happens when you remove the eyeless protein?

Eyes will not form

What is the eyeless protein required for? What is it true for?

Formation of eyes and true for flies and humans

What happens when you remove the white protein?

Eyes will be white

What happens when you mutate the white protein?

It cannot make the white protein and therefore, the eyes will not have color

What is the white protein required for?

Pigment in the eyes

What happens if you remove mRNA?

The protein is not made

What is hybridization?

It is the process of combining 2 complementary single-stranded DNA or RNA molecules and allowing them to form a single double-stranded molecule through base pairing.

How many strands does DNA have?

2 strands

Is the sequence of one strand complementary to another strand in DNA?

Yes it is

What does complementary mean?

The bases present on one strand can bind to the bases on the other strand

What does A bind to?

T

What does C bind to?

G

Can RNA strands hybridize?

Yes they can

What does hybridization of RNA allow us to do?

To detect mRNA molecules

What are oligonucleotides?

They are small nucleotides (small RNA molecules) that are complementary to your RNA.

Describe how oligonucleotides can be used to detect mRNA molecules

1. Oligonucleotides are conjugated to fluorphores

2. Inject the oligonucleotide into a cell

3. It will hybridize to the complementary RNA

4. You can determine where mRNA is based on where it is glowing

What can you block by using an oligonucleotide? Does this idea actually work?

You can block translation due to the oligonucleotide hybridizing to the complementary region of mRNA. No it does not

What did Andrew Fire and Craig Mello do?

They injected anti-sense RNA into c.elegans to remove the protein that it was complementary to.

What specific organism were Andrew Fire and Craig Mello working on?

A mutant of c. elegans that would twitch when the specific protein was removed.

Describe the outcome of the experiment of Andrew Fire and Craig Mello

Injected sense RNA and hoped nothing would happen because sense RNA is the same strand. Nothing happened. Injected anti-sense RNA and expected to see twitching. Nothing happened. Injected dsRNA and it gave the twitching phenotype.

What does injection of dsRNA inhibit?

Synthesis of specific proteins

What type of protein do c.elegans have?

A GFP

What happens specifically when dsRNA is injected into c.elegans?

The GFP has a complementary sequence to the injected dsRNA and therefore the GFP will not be made anymore

What does injection of dsRNA into a cell promote?

Degradation of the mRNA that it is complementary to

What is RNA interference (RNAi) a mechanism of?

Gene regulation

Describe the process of RNAi

1. Inject cell with dsRNA

2. Dicer recognizes the dsRNA and will trap it and cleave it into smaller dsRNA pieces

3. One of the pieces is given to the RISC complex

4. RISC will go and find other mRNA that is complementary to the small piece it has

5. It will degrade the mRNA molecules

Where is the RISC complex found?

In the cytoplasm

Why does dicer recognize dsRNA?

This is because it is not normally present in the cell

What is RISC?

It is a nuclease that will degrade mRNA but only the ones it is complementary to

What is dicer?

It is a nuclease enzyme that cleaves dsRNA into small fragments

What are the small fragments of dsRNA called?

siRNA

What does siRNA stand for?

Small interfering RNAs

Approximately how many base pairs are the siRNAs?

23 nucleotide pairs

Are the siRNAs directly passed to the RISC complex?

Yes they are