RR14: Post-transcriptional gene silencing

Question 1

What happens when we introduce double-stranded RNA in a given gene in an organism?

Answer 1

It would wipe out all the mRNA corresponding to that gene sequence.

Question 2

How can we get double-stranded RNA in C. elegans?

Answer 2

You introduce the double-stranded RNA in a bacteria.
Then, you feed the bacteria to C. elegans.
It gives us the mutant phenotype.

Question 3

Would introducing double-stranded RNA in humans work to modify the phenotype?

Answer 3

Yes.

Question 4

How can you create a Drosophila fly with white eyes instead of red?

Answer 4

1. Introduce a transgene in the fly that makes dsRNA that corresponds to the gene that gives red eyes.
2. The dsRNA generated from the transgene will eliminate the mRNAs that are homologous with it
3. Since we made sure the transgene would produce dsRNA corresponding to the gene that gives red eyes, we end up with a phenotype that doesn’t have red eyes.

Question 5

How can you increase the number of flowers that plant can grow?

Answer 5

1. Introduce transgene that makes dsRNA that corresponds to the Clavata gene.
2. The dsRNA will eliminate the mRNAs that are homologous with it
3. It will eliminate the expression of gene Clavata
4. By eliminating the gene that limits the amount of flowers a plant produces, we can now have way more flowers growing from the plant.

Question 6

What’s RNAi, using siRNAs?

Answer 6

RNA Interference.
We use dsRNA to give rise to siRNAs that have a 100% complementary with their target mRNA.

Question 7

Why would we use siRNAs over miRNAs?

Answer 7

The siRNAs are cleaving and degrading the mRNA transcripts that are 100% complementary to them.
The miRNAs are only blocking translation or destabilizing the mRNA target, they’re not destroying them forever.

Question 8

What happens when the dsRNA made form a transgene comes into the cell?

Answer 8

1. Interacts with Dicer
2. Dicer chops the dsRNA into siRNA
3. Dicer gives the siRNA to RISC.
4. The Argonaute protein in RISC unwinds the dssiRNA with ATP hydrolysis.
5. The RISC will use one of the strands of the siRNA to get to its mRNA targets that have antisense homology to the siRNA (100% perfect pairing).
6. Argonaute protein recongnizes the mRNA that has 100% complementary with the siRNA and it will cut the mRNA.
7. mRNA cut in the middle = kiss of death = endonucleolytic cleavage done by the slicer activity in the Argonaute protein
8. It eliminates the mRNA by the 3’ to 5’ and 5’ to 3’ decay

Question 9

Can a single siRNA target more than 1 mRNA?

Answer 9

Yes. As long as the sequences are 100% complementary, the siRNA can be reused to eliminate an entire mRNA family.

Question 10

Why are centromeres in Palm B yeast silenced?

Answer 10

We found dsRNA that correspond to the sequences in the centromeres.
It gives rise to siRNAs that interact with those regions and recruit chromatin-modifying proteins to shut down the centromeric regions of yeast.

Question 11

What does it mean that siRNAs are involved in silencing the centromeric regions of yeast?

Answer 11

It means that there is a nuclear kind of RNAi that mediates chromatin changes.
siRNA involved in silencing centromeric regions in Palm B are not destroying mRNA targets, they’re just changing the configuration of the chromatin.

Question 12

Apart from recognizing and destroying mRNAs, what is another role of siRNAs?

Answer 12

They can recruit major chromatin-modifying complexes to shut down regions of the genome.

Question 13

What are piRNAs?

Answer 13

They are another class of small RNAs that interact with PIWI proteins.

Question 14

What is a PIWI protein?

Answer 14

It’s a type of Argonaute protein.
It interacts with piRNAs.

Question 15

What are the role of piRNAs and PIWI proteins?

Answer 15

It’s to protect the germ cells.

Question 16

What are germ cells?

Answer 16

They are the cells that give rise to gametes.

Question 17

How are piRNAs made?

Answer 17

piRNAs are transcribed from a DNA cluster made up of integrated disabled transposable elements in flies.
1. ds piRNA cluster of disabled transposons
2. RNA pol 2 goes in that piRNA and creates a piRNA precursor that has sequences shared with transposons.
3. PIWI protein meets the piRNA precursor and they form a ribonuclear protein complex
4. PIWI will seek out RNAs that are complementary to the piRNA sequence, which might be transposons that got transcribed.
5. Transposons that got transcribed into mRNA can cause grief to the cell because it can give rise to truncated proteins.
6. piRNA + PIWI protein interact with the mRNA
7. PIWI protein has a slicer activity and will cut that transposon mRNA into 2 sections.
8. One of the sections of the transposons mRNA can be used to make more piRNA.
9. That way, they’re protecting the germ cells from these transcribed transposable element DNA sequences.

Question 18

What are the functions of piRNAs?

Answer 18

Protect the germ cells
Nuclear function: assemble chromatin-modifying complexes to shut down specific regions of the genome (like in the centromere of flies)

Question 19

Why would we want to use the nuclear function of piRNA?

Answer 19

piRNA assemble chromatin-modifying complexes to shut down regions of the genome.
It can be important to recognize genes that come from the self and genes that come from the non-self, like virus and transposable elements.

Question 20

Do piRNA and PIWI protein complexes require Dicer?

Answer 20

No. Even if they’re smaller than miRNAs and siRNAs, they don’t need Dicer.

Question 21

How can we get rid of viruses when they come in?

Answer 21

The cell will use RNAi to attack invaders. A ds viral RNA is generated from RNAi and it can chop the virus into little pieces.

Question 22

How do viruses protect themselves from being attacked by dsRNA that will come and cut them?

Answer 22

1. Viruses will sequester all of their RNA products to protect them from the dsRNA of the host cell.
   They sequester their RNA product by generating non-coding RNAs that have complementary sequences to miRNA or siRNA that will come in and interfere with the virus.
2. They use circular RNAs to soak up the important miRNAs that can come and interfere with the virus.

Question 23

What’s dosage compensation?

Answer 23

In mammals, it’s when the cell has to dose the amount of X chromosome present. With a chromosome-counting mechanism, the cell has to decide which X chromosome is going to be silenced in females.
No need for silencing in males, because they have 1 X and 1 Y chromosomes.

Question 24

During dosage compensation, do all the cells in one female mammal choose the same X chromosome to silence?

Answer 24

No. Every cell picks the X chromosome they want.

Question 25

What happens to the silenced X chromosome, when a female cell has to do cell division?

Answer 25

The same chromosome that was inactivated in the mother cell will be inactive in the daughter cells too.

Question 26

How do we call Inactive X chromosomes?

Answer 26

Barr Bodies

Question 27

Why are calico cats different colors?

Answer 27

Because the X chromosome is associated with the colour of the fur. So the fur is representative of the coat color on the active X chromosome.
So, in early embryogenesis, the cells had to decide which X chromosome was activated and with cell division, other cells got the same activated X chromosome, which can create patches because not all the cells picked the same X chromosome in early embryogenesis, so not all the same coat colour.

Question 28

What happens if a female has 4X chromosome in one cell.

Answer 28

The cell has to inactivate 3 X chromosomes and only leave 1 active.

Question 29

What is XIST?

Answer 29

It’s a gene product.
XIST locus encodes a long non-coding RNA (lncRNA)
It binds to discrete regions of the X chromosome. It spreads along the X to extinguish gene expression.

Question 30

What’s the job of XIST locus?

Answer 30

To inactivate X chromosomes to make sure only 1 X chromosome is activated in each cell.

Question 31

Does XIST encode a protein?

Answer 31

No. It encodes a long non-coding RNA.

Question 32

What coats the X chromosome to inactivate it?

Answer 32

It’s the long non-coding RNA encoded by XIST.

Question 33

Who is responsible for making sure that the daughter cells have the same X chromosome that’s inactivated?

Answer 33

It’s the long non-coding RNA encoded by XIST.
The effect of the lncRNA are propagated into the next cell division.

Question 34

What is TSIX?

Answer 34

It’s the antisense of XIST.
It’s a long non-coding RNA that antagonizes XIST expression.
It defines the active chromosome.

Question 35

What causes the random decision of inactivating or activating one of the X chromosomes?

Answer 35

It’s caused by transcriptional interference. One of the lncRNA interferes with the transcription of the other, so one is more expressed than the other.
Whichever one is more expressed will decide if the chromosome is active or inactive.
If the transcription of XIST is higher, then the chromosome will be inactive, and vice versa.

Question 36

When one chromosome is coated with lncRNA of XIST or TSIX, how does the other X chromosome know to be activated or inactivated?

Answer 36

As soon as the initial decision is made, the amount of transcription doesn’t matter and if the first one is activated the other will just be inactivated without a fight.
And then, the decision gets propagated through cell division.

Question 37

How is the decision of the active or inactive X chromosome propagated?

Answer 37

By coating the entire X chromosome that’s inactivated, XIST RNA recruits complexes involved in the modification of the chromatin.
It gives repressive chromatin modifications that condense the X-chromosome and it’s almost inaccessible for transcription factors.
Since the gene expression is inhibited in the inactivated chromosome, the decision is maintained in daughter cells.
The modification of the histones to compact the chromatin are propagated.

Question 38

What are epigenetic processes?

Answer 38

Modifications to DNA that regulate whether genes are turned on of off.

Question 39

What’s HDAC3 enzyme?

Answer 39

It’s a histone deacetylase. It will remove the acetyl groups off the histone tails resulting in a compaction of the chromatin.

Question 40

Is HDAC3 enzyme used during the dosage compensation?

Answer 40

Yes. It’s used by the XIST mRNA that’s coated on the inactive X chromosome.
The XIST mRNA will recruit HDAC3 with other complexes to take off the acetyl groups off the histone tails to compact the chromatin.

Question 41

What is the methylation of Lysine 27 associated with?

Answer 41

It’s associated with compacting of the histones to shut down transcription.