Lecture 26 (RR14): Post-transcripional gene silencing Flashcards
Craig Mello
- Graig Mello was interested in embryonic events, so he was injecting bits of RNA or antisense oligonucleotides that were complementary to the maternal RNAs that are dumped into early embryo to see if he could disrupt some of those early processes to understand better how they were working. He was getting surprising phenotypes (sometimes it was heritable).
- They figured out that when you inject double stranded RNA into the germ line of C elegans, or introduce it even by soaking animals in a solution that contains double stranded RNA and the animals pump in the double stranded RNA through their mouth. It results in the loss of function of that particular gene product and it was really dependent on the double stranded RNA per se.
- In the C. elegans, when they injected double stranded RNA in it. they got a phenotype that corresponds to the loss of function of the gene product of the double stranded RNA that you introduced.
- Craig Mello was working with Victor Ambrose, little did they know that they were actually working on a common pathway where small RNAs (or small non-coding RNAs) were able to elicit major downstream events to control gene expression.
RNA mediated Interfereance
(RNAi)
In the situation of RNA mediated interference (RNAi), these small non-coding RNAs are referred to as small interfering RNAs and the whole process is now believed to be a part of a defense mechanism, that cell have developed in order to defend themselves against viruses and parasites that might make double stranded RNAs if they are plunked into the appropriate position in the genome where they get transcribed in a convergent mechanism.
MicroRNAs
On the other hand, microRNAs are physiological. They are required for normal development.
* The microRNA pathway tends to be regarded as a means of fine tuning gene expression in line with physiological changes and responses as well as development.
What happened when investigators that’d seen what was happening in C Elegans began to try in different organisms?
- In flies, if you can introduce a transgene that would eventually form a snapback RNA (it is going to sense in one direction and then be a little bit of a leader and then allow for antisense RNA that will continue to fold back on itself). This gives rise to a long stem loop. If you can introduce those into drosophila, it will be recognized by the critical enzymes involved in the RNA mediated interference (RNAi) pathway and give rise to the same kind of mutant phenotypes that people were seeing with all the C. elegans.
- Therefore, it is enough to have a double stranded RNA that corresponds to a given gene product, even if you introduce it on a transgene by making a fullback RNA, it is a sufficient trigger to induce the RNAi pathway.
- dsRNA introduced by transgenes or through transfection of modified small dsRNAs (siRNAs) causes loss of function phenotypes that are typical of genetic mutations in the targeted gene.
Works on plants: Example with Clavata 3
and Mammals
It is a gene product that’s required to restrict the stem cell population that will give rise to the floral organs. That way you only have a few flowers that will form from the stems in the terminal regions of plants.
But if you introduce a transgene, that would form a double stranded snap back RNA, you’ll get a phenotype that’s almost the same as if you had a true genetic mutant of clavata 3. That shown here by all these extra stem cell centers that are giving rise to new floral organs that normally should never be allowed.
- But because you are removing that restriction using RNAi, you get rid of all the mRNA. Almost as if it was a genetic mutation where the gene product isn’t made correctly.
You can do the same thing with mammalian organism. The mouse has been treated using RNAi, these small interfering RNAs. They were introduced very early during embryonic development and in this case the siRNAs correspond to a gene product that is known to be mutated in patients that have Tay-sachs disease. In the mouse that emerges from that manipulation, it phenocopies that mutation in the mouse → just as if you had knocked the gene out (except this is much simpler, you can use small non coding RNAss in order to eliminate entire populations of mRNA that correspond to a gene products that you might be interested in).
This has revolutionized how people do research in the laboratory. Suddenly, you could get loss of function phenotypes for just about any gene product that interests you.
microRNA pathway vs RNAi pathway
- Both of these triggers, the microRNA and the double stranded RNA, were converging on one single pathway. That was all mediated by Dicer protein (chopping the initial trigger RNA).
In the microRNA pathway: lin-4 comes from a long precursor (60 nucleotides long) that is transcribed in the nucleus and then processed. Eventually Dicer will use that as a substrate to cut it into microRNAs.
In the RNAi pathway: the same sort of thing is true. A double stranded RNA (from snapback transgene or that you have prepared in lab), these triggers will be recognized immediately by Dicer and it chops them into small 22 ish nucleotide small double stranded RNAs.
THEY USE THE SAME DOWNSTREAM EFFECTORS IN BOTH CASES.
Developmental timing mutants in C. elegans helped to elucidate a new pathway
- In the nematode C. elegans lin-4 lf mutants (lin stands for lineage abnormal) look very similar to lin-14 gf mutants.
- lin-4 encodes a small RNA that has considerable homology (antisense) to regions of the lin-14 3’ UTR.
- lin-4 RNA is synthesized as a longer precursor than it has to be processed, after which it binds its target sites and affects translation of the lin-14 mRNA.
- Argonaute proteins, a component of RISC, and Dicer are both involved in this pathway.
- This process is developmentally or physiologically regulated
RNAi steps
1) Dicer is an RNAse III like enzyme. It will chop the dsRNA into siRNAs (if it is coming from the RNAi pathway) or microRNA. It chops them so that they have a little 3’ overhang.
2) Dicer will hand off the double stranded RNA that it processed to a large protein called an RNA induced silencing complex (RISC).
- It acts as a dimer to cleave dsRNA into 21-23nt fragments that are then bound by an Argonaute protein in the RISC.
- The microRNA pathway uses a specific group of Argonautes that participate in a microRNA RISC complex (MI RISC).
3) ATP hydrolysis drives the unwinding (helicase) activity of RISC .
4) The RISC uses the ssRNA product as a guide strand to target the complex to complementary cellular RNAs, finally associating through Watson-Crick base pairing. The guide strand (with the RISC and Argonaute protein) will be escorted to its final target (it will try to base pair with a number of targets until it finally reaches the appropriate one)
5) If there is 100% complementarity or near 100% complementarity between the guide RNA and its target. A specific activity within the argonaute protein called Slicer will cut the mRNA target with an endonucleolytic cleavage → kiss of death…
6) The resulting cleaved transcripts are then degraded by cytoplasmic ribonucleases
MicroRNA steps
- The same kind of thing takes place with the microRNA pathway (as the RNAi pathway), the Argonautes are a little specific to microRNAs.
1) A single guide strand will escort the RISC to its final target. In this case the complementarity cannot be 100% (if you look at the Lin-4/Lin-14 targets then you will notice that there is a section that bulges out). It is never 100% complementary → signature of microRNA.
2) The microRNA along with the risk will interact with its targets and because of the imperfect complementarity, it will affect the stability of the RNA or block translation.
3) The slicer activity of the Argonaute protein won’t cut the mRNA in the middle. This will affect the adenylation or block translation directly (but there is no slicing). - Most people believe these days that the microRNA pathway is required to fine tune specific gene expression during development because theoretically you can reuse these mRNAs if they are not totally degraded following adenylation.
RNAi vs microRNA Roles
- One is really for defense against RNA based aggressors (RNAi) and the other is really important for developmental or physiological adjustments (microRNA).
- So the difference between the two is:
→ If you stop the RNAi pathway in an organism, they would probably survive, but they wouldn’t defend themselves well (and maybe there is going to be noise due to transposons).
→ If you disrupt components of the microRNA biogenesis pathways, the animals die very early during embryogenesis. It is ABSOLUTELY critical for development.
RNAi process vs microRNA process
BOTH ARE TRIGGERED BY A DOUBLE STRANDED RNA MOLECULE (dsRNA).
Although there are differences between the RISC complexes between the 2 pathways one of the major distinctions to remember is that microRNA don’t work in 100% complementarity. They always require that they are NOT 100% complementary to their targets and this will lead to destabilization or direct translational regulation.
Whereas, siRNAs that are working through the RNAi pathway use 100% complementarity and destroy their mRNA targets by endonucleolytic cleavage that is mediated by the slicer activity of the Argonaute protein in the RISC complex. This leads to rapid degradation mediated by the degradation pathway that works in both polarities.
Other non-coding RNAs nucleate complexes that trigger chromosomal silencing
- There are a number of dsRNAs that seem to be present and are required for changes to the chromatin that correspond to the DNA sequences around these particular regions of the centromere.
- Centromeric regions are associated with the kinetochore - a structure that builds around the centromeres and is very important for accurate cell division. Chromatin within the centromeres must be silenced. It is important that not much is going on there because presumably you need something relatively stable to set up this structure.
- In S. pombe (fishing yeast) small RNAs are required for this silencing mechanism.
- Investigators identified small dsRNAs that correspond to the DNA sequences around the centromere. They also found that Dicer is required for this silencing that gives rise to heterochromatin formation. A number of other proteins mediate the recruitment of chromatin modifying enzymes that put down H3K9 trimethylation marks. These marks typical of chromatin condensation, thereby closing down all the chromatin to transcription.
- **dsRNA nucleates a complex that involves several proteins that are all involved in generating H3K9me3 **
- A similar mechanism functions in vertebrates to silence centromeric regions. Regions of heterochromatin somehow depended on the role of Dicer and again small dsRNA segments.
- It appears that although Dicer and these small dsRNAs, whether it be microRNA or siRNAs, play a very important role in:
→ regulating developmental events (with microRNAs)
→ defence against genetic intrusions (in the case of RNAi)
→ They also seem to be important in the formation of structures that give rise to changes in the chromatin that eventually will affect gene expression.
piRNAs and PIWI proteins
- piRNAs and PIWI proteins protect the germ lines of most animals
- piRNAs are transcribed from a DNA cluster made up of integrated of disabled transposable elements in flies.
- Their function does not require Dicer (they are in fact transcribed) but does require an **Argonaute protein called PIWI. **
- PIWI is the piRNA sort of Argonaute → it is the thing that will carry out the downstream events that have been instructed by the PIWI or piRNAs.
- Not all small non-coding RNAs are dicer dependent or work in a manner that is related to microRNAs or RNAi (the group of piRNAs).
- Every organism that uses a piRNA based method of gene regulation kind of does it in a different way. - They are modified and then bind to their targets through antisense complementarity and PIWI will then cleave (slice) the transposon RNA.
- They are involved in many processes related to gene expression from regulating mRNA stability to enhancing protein synthesis.
piRNAs in the fly
- Recognize transposon products.
- Slice the RNAs and therefore protect the cell. They are very important for protecting germ cells in most organisms.
piRNAs in C elegans process.
- There is a cluster with a number of independent mini genes, all of which encode different piRNAs.
- They are all transcribed independently so you have tons of these small piRNAs that are floating around the cell.
- They bind to their cognate, PIWI like Argonaute complexes, and then interact with specific regions of the genome which are either self or non self.
- It gets complicated at that point, there are other argonaute proteins that define self, particularly in the germ cells, so the germ cells know which gene should be expressed within the germ cells.
- All the other sequences that don’t seem to be related to normal germ cell function are bound by piRNAs and C elegans and then they are degraded (or heterochromatin so that the gene doesn’t get expressed).
Calico cats
- The patched nature of the fur of calico cats/tortoise shell coloured cats indicate regions where the inactivation of the X chromosome was altered, therefore providing coat colour information from two different X chromosomes.
- Coat colour is on the X chromosome. If you extinguish one X chromosome you get one color, if you extinguish the other you get a different coat colour.
- What you end up seeing is a manifestation in an animal of all of the clones of cells that made that initial decision and gave rise to the coat of the adult mammal.
- All the female cats are mosaic.
- You can see the level of gene expression by staining with antibodies that recognize clear marks that associate with transcriptional activation. The only chromosome that is strained red is because there is no transcription.
XIST
XIST is a seXIST gene product…
How do you make the first decision to inactivate an X chromosome?
It is all dependent on this long non coding RNA called Xsit:
* The XIST locus encodes a long non-coding RNA (lncRNA)
* It binds to discrete regions of the X chromosome and spreads along the X to eventually X-tinguish gene expression.
- It expresses on the presumptive inactive X chromosome and coats the entire chromosome (the red covering on the image).
- Once it covers the X chromosome one time then every other cell division thereafter does not require Xist anymore.
- The chromatin is modified in such a way that it knows that that particular X chromosome must be condensed and transcription must be off.
* Although many mechanisms have been proposed it is still not fully clear how this RNA functions and what is involved in its activation or specificity.
Long non -coding RNAs (lncRNAs)
lncRNAs can regulate gene expression
* In early embryogenesis, both TSIX and XIST are expressed in female cells.
* The random choice to inactivate the X chromosome in female mammals is a result of a stochastic decision to express XIST or not.
- The inactive X will occur on the X chromosome that has more XIST that is being expressed with respect to TSIX. TSIX ensures that the other active X chromosome never becomes inactivated.
* TSIX expression biases against XIST and the two are generally mutually exclusive later in embryonic development → battle between 2 long non-coding RNAs.
- TSIX is just XIST backwards because it is an antisense RNA that gets transcribed in the other direction in the same locus → there is some antagonism between the two promoters.
REMEMBER:
- XIST is a long non-coding RNA (lncRNA) that coats the X chromosome and silences it
- TSIX is a long non-coding RNA (lncRNA) that antagonizes XIST expression
XIST RNA recruits a chromatin modifying complex
- By coating the entire X chromosome in cis XIST RNA it recruits chromatin modifying complexes.
- HDAC → histone deacetylase complex. It will remove the acetyl groups off histone tails and condense the chromatin.
- Polycomb Group (PcG) comes in and adds all of these K27 methylations to condense the chromatin and block transcription around that region. Thereby, inactivating gene expression of the inactive X chromosome.
- Polycomb group (PcG) proteins are epigenetic regulators of transcription that have key roles in stem-cell identity, differentiation and disease. - This results in repressive chromatin modifications that condense the X- chromosome and render it mostly inaccessible to transcription factors
- Gene expression is largely X-tinguished from the inactive X and this decision is maintained thereafter in every new daughter cell through epigenetic processes
