RR14: Post-transcriptional gene silencing Flashcards
What happens when we introduce double-stranded RNA in a given gene in an organism?
It would wipe out all the mRNA corresponding to that gene sequence.
How can we get double-stranded RNA in C. elegans?
You introduce the double-stranded RNA in a bacteria.
Then, you feed the bacteria to C. elegans.
It gives us the mutant phenotype.
Would introducing double-stranded RNA in humans work to modify the phenotype?
Yes.
How can you create a Drosophila fly with white eyes instead of red?
- Introduce a transgene in the fly that makes dsRNA that corresponds to the gene that gives red eyes.
- The dsRNA generated from the transgene will eliminate the mRNAs that are homologous with it
- 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.
How can you increase the number of flowers that plant can grow?
- Introduce transgene that makes dsRNA that corresponds to the Clavata gene.
- The dsRNA will eliminate the mRNAs that are homologous with it
- It will eliminate the expression of gene Clavata
- By eliminating the gene that limits the amount of flowers a plant produces, we can now have way more flowers growing from the plant.
What’s RNAi, using siRNAs?
RNA Interference.
We use dsRNA to give rise to siRNAs that have a 100% complementary with their target mRNA.
Why would we use siRNAs over miRNAs?
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.
What happens when the dsRNA made form a transgene comes into the cell?
- Interacts with Dicer
- Dicer chops the dsRNA into siRNA
- Dicer gives the siRNA to RISC.
- The Argonaute protein in RISC unwinds the dssiRNA with ATP hydrolysis.
- 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).
- Argonaute protein recongnizes the mRNA that has 100% complementary with the siRNA and it will cut the mRNA.
- mRNA cut in the middle = kiss of death = endonucleolytic cleavage done by the slicer activity in the Argonaute protein
- It eliminates the mRNA by the 3’ to 5’ and 5’ to 3’ decay
Can a single siRNA target more than 1 mRNA?
Yes. As long as the sequences are 100% complementary, the siRNA can be reused to eliminate an entire mRNA family.
Why are centromeres in Palm B yeast silenced?
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.
What does it mean that siRNAs are involved in silencing the centromeric regions of yeast?
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.
Apart from recognizing and destroying mRNAs, what is another role of siRNAs?
They can recruit major chromatin-modifying complexes to shut down regions of the genome.
What are piRNAs?
They are another class of small RNAs that interact with PIWI proteins.
What is a PIWI protein?
It’s a type of Argonaute protein.
It interacts with piRNAs.
What are the role of piRNAs and PIWI proteins?
It’s to protect the germ cells.
What are germ cells?
They are the cells that give rise to gametes.
How are piRNAs made?
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.
What are the functions of piRNAs?
Protect the germ cells
Nuclear function: assemble chromatin-modifying complexes to shut down specific regions of the genome (like in the centromere of flies)
Why would we want to use the nuclear function of piRNA?
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.
Do piRNA and PIWI protein complexes require Dicer?
No. Even if they’re smaller than miRNAs and siRNAs, they don’t need Dicer.
How can we get rid of viruses when they come in?
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.
How do viruses protect themselves from being attacked by dsRNA that will come and cut them?
- 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. - They use circular RNAs to soak up the important miRNAs that can come and interfere with the virus.
What’s dosage compensation?
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.
During dosage compensation, do all the cells in one female mammal choose the same X chromosome to silence?
No. Every cell picks the X chromosome they want.
