RR14: Post Transcriptional Gene Silencing Emerging Roles for RNAs

Question 1

what happens when you inject dsRNA to any given gene in a worm? (C. elegans)

Answer 1

all the mRNAs corresponding to that gene sequences would be wiped out

Question 1

what are the ways in which dsRNA can be introduced to the C.elegans worm?

Answer 1

inject it anywhere in the worm, soak the animal in it, introduce constructs into bacteria that would make dsRNA in their own cells and feed the bacteria to c elegans

Question 2

what is RNA mediated interference?

Answer 2

introducing small dsRNAs to shut off some genes

Question 3

how can flies with white eyes be made?

Answer 3

• red eyes is the wild type
• If you introduce a Trans gene that makes one of those snap back dsRNAs that correspond to the gene that gives flies red eyes, you end up getting a white eye phenotype
• DsRNAs eliminate the mRNAs that tell the eyes to be red

Question 4

how can you grow more shoots and flowers in a plant?

Answer 4

• Same is true for the plants: CLV3 limits the stem cell population In plants so that you only grow a few shoots and flowers
• If you get rid of CLV3 using RNA mediated interference (RNAi) you end up getting whopping numbers of these new structures normally limited by the function of that gene

Question 5

how can RNAi be used to introduce a disease in mice?

Answer 5

by using RNAi based mechanisms, you can introduce specific dsRNAs that will eliminate that gene product for hexosaminidase A you can give the mouse tray sachs disease

Question 6

what can we determine by eliminating certain genes?

Answer 6

exactly what they do in physiology and their role in development

Question 7

what is the RNAi process? (steps)

Answer 7

• Works in a manner similar to the miRNA pathway
• dsRNA comes into the cell it’ll be met by dicer (endoribonuclease) that will cut it down to create small interfering RNAs (siRNAs) That have characteristic overhangs
• they dicer hands it off to the RISC complex, and at the core is the argonaute protein
• The argonaute protein will recognise what they have to do based on the dsRNAs that they have to interact with
• MiRNA specific argonaute RISC complexes, and RNAi specific RISC complexes, depending on the argonaute protein at the core
• The RISC complex using ATP helicase will use one of those strands as a guide
• the RISC complex will accompany the guide to its final target where it will have 100% homology
• When the RISC complex recognizes that 100% complimentarity, it will cut the mRNA in the middle (kiss of death)
• Endonucleolytic cleavage, gets chewed up very rapidly
• doesn’t just block the translation but eliminated the entire mRNA, you can wipe out entire families based on homology

Question 8

what are the differences between the RNAi vs miRNA processes?

Answer 8

• the distinguishing features between the two pathways lies in the ability of those guide RNAs (miRNA) to only interact with their targets with limited homology vs the siRNAs that interact with 100% complimentarity
• The outputs are very different
• MiRNA: destabilizing or block translation
• SiRNA (RNAi): eliminates the mRNA through endoribonucleotlytic cleavage, slicer activity within the argonaute protein

Question 9

what do both pathways start with? (RNAi and miRNA)

Answer 9

• Both pathways start with a dsRNA trigger/precursor

Question 10

how can centromeres be silenced in pombe yeast?

Answer 10

• There are dsRNAs that can be isolated that correspond to sequences within the centromeres
• Those ds siRNAs interact with those regions and recruit a host of chromatin modifying proteins to shut down the centromere region of yeast in pombe

Question 11

how can dsRNA play a role in shutting down regions of the genome?

Answer 11

by modifying the chromatin

Question 12

what is the role of piRNAs and what do they associate with?

Answer 12

interact with PIWI (an argonaute protein)
their role is to protect germ cells

Question 13

why are germ cells important?

Answer 13

• Germ cells are important because they give rise to gametes and are considered the immortal lineage that goes on into the future

Question 14

how are piRNAs generated? what is the whole process of protection?

Answer 14

• In flies, piRNAs are generated from a cluster found on the chromosome
• Within that cluster found that there are old sequences of transposable elements all jammed together
• When RNA pol II goes through that region it makes a long RNA (piRNA precursor) that corresponds to a sequence/opposite sequence present in transposable elements
• These are then met by another argonaute protein (PIWI) and they form a ribonucleoprotein complex, where PIWI and associated piRNA seek out RNAs that are complimentary to that sequence, which might be transposable elements that actually got transcribed that can cause grief in the cell (truncated proteins or other weird translated products)
• in order to limit that, piRNA associated with PIWI will interact and the PIWI has a slicer activity as well, and it will cut that transposons mRNA into two sections, and then one of the sections can get reused to go back and be involved in an amplification process to make more of the piRNA PIWI complexes
• protect germ cells from these transcribed transposable element DNA sequences that cause damage

Question 15

what can piRNAs also do besides germ cell protection? (2 more)

Answer 15

also have nuclear functions so that they can assemble chromatin modifying complexes to shut down regions of the genome
important in recognizing genes that come from self vs genes that come from non self (viral invaders or transposable elements or other)

Question 16

what do piRNAs do not need compared to other small RNAs?

Answer 16

do not need dicer, transcribed and processed by another mechanism

Question 17

how do viruses adapt to the dsRNAs sent by the cell as an immune response?

Answer 17

viruses can sequester these small RNAs by generating long non coding RNAs that have homologous/complimmentary sequence to the critical miRNA or siRNA that could be involved in interefering with its infection
* Sometimes use circRNA or sponge RNA so that they can titrate all of the important microRNAs that can induce an immune response response that would eliminate the virus

Question 18

how can cells defend themselves against viruses?

Answer 18

cells use RNAi pathways to defend themselves
use these small RNAs to get rid of RNA based products from the virus

Question 19

what happens in female in early embryogenesis?

Answer 19

very early in mammalian embryogenesis, all the cells that made up the embryo had to make the decision based on a chromosome counting mechanism to silence one X chromosome
* One dose of the X chromosome is sufficient
* random decision that has to take place around the 100 cell stage (very early)
* Every cell does it its own way, and every single division after that, the same X chromosome inactivated in the mother cell will be inactivated thereafter

Question 20

how can inactive X chromosomes be visualised in micrographs?

Answer 20

can be seen as Barr bodies, because they are very densely compacted

Question 21

what gene product silences the X chromosome?

Answer 21

XIST

Question 22

where is XIST active?

Answer 22

female mammalian cells

Question 23

what does XIST do in those early embryogenesis stages?

Answer 23

• During those early stages, XIST is expressed and it encodes a long non coding RNA (not a protein)
• Pol II transcript that is polyadenylated spliced and everything and then it coats the entire X chromosome, and by doing that it is able to extinguish gene expression off that inactive X

Question 24

what is transcriptional interference?

Answer 24

transcription of one interferes with the other, shifts the balance

Question 25

what role does transcriptional interference have in making the decision for X chromosome silencing?

Answer 25

• another long non coding RNA called TSIX which is the antisense of XIST interferes with XIST function
• increase of one transcription over the other (TSIX vs XIST) will define whether or not the chromosome stays active or not
• If TSIX is higher than XIST, that chromosome remains active and vice versa
• once XIST has coated the chromosome and shut it down, it is no longer required
• Decision is made and propagated

Question 26

what happens when XIST coats the chromosome? (how does it get silenced)

Answer 26

• recruitement of several multi protein complexes involved in compacting the DNA by XIST expression
• interaction between XIST and RNA binding protein (SHARP)
• Form condensates around the chromosome that is being inactivated = RNA acts like glue
• brings in HDAC3 enzyme, histone deacetylase
• Removes acetyl groups, compaction of the chromatin
• Particularly histone 3 and lysine 27 (H3K27), shutting down
• Form repressor heterochromatin complex
• Extinguish a great deal of the gene expression on that inactive X