Lecture 25 (RR13): Post-transcriptional/translational regulation of gene expression Flashcards
RNA surveillance and quality control
- SR proteins define the exons so introns can be appropriately excised.
- Polyadenylation of the pre-mRNA
- Export factors are loaded onto the mRNA
- All remaining factors must be removed in the pioneering round of translation- if not… NMD nonsense mediated decay
When does the RNA Surveillance process become important?
The RNA Surveillance process becomes important when mRNAs are altered or they’re mutants such that they give rise to inframe stops that happen very early or prematurely in the mRNA.
Inframe stops
- An inframe stop can be problematic in many ways. One of the major issues that could arise from this is that it could make a truncated protein. It will only make ½, ¼, ⅓…of a protein.
→ This can give rise to dominant negative variance of a given protein. These wreak havoc in the cell.
→ Dominant negative proteins almost act like mutant proteins, they can do half the job of a normal protein - but not the more important half.
Ex: you make a dominant negative variant of a sex steroid hormone receptor that will still bind the steroid but cannot activate the appropriate genes downstream. By doing so, you stop up all of the steroids that are present in the cell but then don’t activate the appropriate genes downstream.
What happens if there is an inframe stop?
- If there is an inframe stop in an mRNA, you will remove that mRNA and the way this is done is through quality control mechanism referred to as nonsense mediated decay (NMD)
- NMD relies on an interesting property that is associated with the first round of translation.
→ Pioneering round of translation: the first ribosomes that are going to transit through an mRNA. At the same time as they are reading that mRNA (the ribosomes), they will kick off any other protein that is associated with the mRNA, including any nuclear proteins that stuck around (ex: SR protein). - When the ribosomes get to the stop, ribosomes dissociates and other ribosomes go whippin through in order to give rise to lots of protein.
- The Case where there is an inframe stop in the middle of that mRNA and the ribosomes arrive at that stop and dissociate. Because they dissociate prematurely they leave a number of proteins on the mRNA (3’ to where that stop is) that would normally be knocked off in the pioneering round. These kind of protein complexes, mRNA or RNA complexes are recognized by NMD machinery and the mRNAs are destroyed.
- If the proteins aren’t kicked off by that ribosome in the pioneering round. Then that could trigger nonsense mediated decay.
- This type of quality control is used by the cell to ensure that things like truncated proteins aren’t generated from variations in the mRNAs.
Stability of cytoplasmic mRNAs
- Degradation of the mRNA takes place by evoked enzymes that chew up the RNAs.
- This means that the stability or degradation of the mRNAs are regulated in a very tight manner.
- This table shows how the stability of mRNAs changes dramatically among organisms: Bacteria, yeast and human cells
- All to say that something is regulating the stabilities of RNAs in the cell.
Eukaryotic nRNAs can be destabilized by a sequence motif
* Many short-lived mRNAs in eukaryotes contain multiple copies of the sequence **AUUUA in their 3’ UTR** (this sequence is responsible for the instability of the mRNA).
* Adding such sequence motifs to the 3’ UTR of a gene that usually does not contain them dramatically destabilizes the hybrid mRNA.
Does RNA decay occur at one end or both ends?
RNA decay can occur at both ends
* RNA degradation can take place in a deadenylation-dependent or -independent manner
* Decapping and 5’-3’ RNA degradation occur most prevalently in devoted sectors of the cytoplasm called P-bodies
Steps to RNA degradation
1) When the proteins interact with the sequence it recruits in an RNA degradation complex. Now, RNA degradation requires the activity of a number of enzymes.
2) It will use a ribonuclease that is going to act either at the 3’ end to chew down the RNA in a 3’ —> 5’ direction or vice-versa.
→ In mammalian cells, the prominent degradation pathway goes from 3’ to 5’ . This is carried out by a large complex called the exosome. The exosome will chew up the RNA as an exoribonuclease (1 nucleotide at a time).
3) This is preceded by an enzyme that will remove the adenosine in the polyA tail, this enzyme is referred to the deadenylase complex. It chews down the poly A tail down to a point that the exosome can begin to act and chew up the RNAs.
→ The deadenylation that takes place can also affect the activity of the degradation enzymes that will work in a 5’ to 3’ direction. This is particularly true when the mRNAs that were being translated and found themselves in circles.
The deadenylase complex will chew down the poly A tail to a point that does not have enough of a platform for poly A binding protein cytoplasmic version to sit down on it. Once the platform is gone, it can no longer make the loop with the 5’ end of the mRNA. If you do not have that protection of the remaining Poly A tail and you don’t have the loop protecting the 5’ end. THe mRNA becomes vulnerable on both sides.
4) Decapping enzyme will remove that 7 methyl guanine cap, thereby exposing the 5 prime terminus of the mRNA to ribonucleases that will chew it in a 5’ to 3’ direction: this enzyme is called XRN1.
5) Exosome is moving in one direction and XRN1 moving in opposite direction.
How does the exosome (exonucleus) work and endonucleus ?
- RNA is threaded 3’-5’ into a complex of similar polypeptides that form a barrel-like structure.
- The exonuclease activity is present at the end of the channel, while an endonuclease activity is present near the exit in case the exonuclease activity fails.
- Exosome has all of these subunits that come together and are more or less repeating to give rise to a long internal channel into which RNA gets threaded and it gets linearlized by an RNA helicase. When it gets to the very end, the exo ribonuclease activity resides and it chews up the RNA coming through the channel one nucleotide at the time.
- If this exosome’s activity is compromised, there is a backup plan. There is a endoribonuclease activity present at the end and so if the RNA actually emerges (which it shouldn’t) it gets attacked by the endoribonuclease activity that snips it in the middle. This is the kiss of death because suddenly those enzymes can work on both ends.
What happens if RNAs are first cleaved in the middle?
- Sometimes RNAs are first cleaved in the middle and then degraded simultaneously by both exonucleolytic degradation pathways ie…5’-3’ and 3’-5’).
- It can work at both ends. The exosome can immediately start to attack the 3’ end of one portion of the RNA and it will degrade it in a 3’ to 5’ end and then XRN1 will degrade the other part of the RNA that arrises from this reaction in a 5’ to 3’ manner.
The stability of some RNAs can be regulated
- The regulation of the mRNA stability is important for cellular homeostasis to ensure that gene expression is controlled at the level or their stability.
- The stability of the mammalian transferrin receptor TfR (which is needed for the import of iron into the cell) is regulated in response to intracellular iron concentration.
Iron Response Element Binding Protein (IRE-BP)
Iron Response Element Binding Protein (IRE-BP) has two alternative, iron concentration-dependent conformations!
* This is a classic example of how an RNA binding protein affects substrates so that cellular homeostasis can be maintained.
*IRE-BP in high iron conditions: *
* it is in an inactive conformation.
* It can’t bind RNA.
In low iron conditions:
* it takes on an active conformation
* capable of interaction with specific RNA elements referred to Iron response elements (IRE).
It plays an important role in iron homeostasis:
- Iron can become toxic and kill the cell once it surpasses certain threshold levels intracellularly.
- This is why the intracellular concentrations of iron have to be critically and exquisitely maintained.
What is responsible for getting iron into the cell?
- The transferrin receptor (TrF) is responsible for getting iron into the cell. It can import iron so that the intracellular levels of iron are appropriate for the cellular function.
- The transferrin receptor mRNA has a number of iron response elements (IRE) in the 3’ untranslated region (3’ UTR). These IREs are adjacent to a number of AU rich elements (AU rich elements are associated with RNA instability).
In high iron situation:
* You do not want a lot of iron going into the cell. Therefore, you do not need a transferrin receptor.
* The mRNA is degraded very rapidly for transferrin receptor because of these enriched AU elements.
However, if the conditions change such that the intracellular concentration of iron decrease below a threshold level, the IRE-BP will take on a conformational change and become active.
- In its active form it will interact specifically with the IREs that are present on the stem loops in the 3’ untranslated region of the transferrin receptor mRNA
- In interacting with its IREs, it blocks any proteins from interactions with those AU rich elements that would otherwise recruit in the RNA degradations complex (exosome).
- Therefore, it stabilizes the mRNA (through the protein interaction) and you end up getting more protein made and there is more transferrin receptor to take iron into the cell.
- It is one mean of ensuring appropriate intracellular iron levels - dependent on playing or regulating the stability of given mRNAs based on these particular RNA binding elements.
mRNA levels and protein levels that do not correlate: Translational Regulation
- Translational regulation is a type of post translational regulation.
- The synthesis of some polypeptides is under strict regulatory control.
- mRNA abundance almost always reflects protein levels such that: more mRNA = more Protein
- When this relationship appears skewed it may indicate that protein synthesis or the stability of the protein is regulated.
- Post transcriptional regulation does not always work through stabilization or destabilization of mRNA products. It can actually work on regulating how well a given mRNA can be translated (translational regulation).
Inhibition of mRNA translation has a pivotal role in Drosophila embryo development
- A number of gene products have been identified in Drosophila melanogaster that are important to make anterior structures and an equal number of gene products that are important for making posterior structures.
- Two of these gene products are shown below:
1) Hunchback is supposed to give rise to these anterior structures.
2) Nanos gives rise to posterior structures.
The nanos RNA is tightly localized at the posterior of the growing embryo and is linked there through its interaction with a few other important gene products. Nanos is concentrated and nicely formed in a crescent at the posterior end.
On the other hand, the hunchback mRNA is also present in a crescent at the anterior end, despite the fact that it’s mRNAs go all through the Embryo. This suggests that the mRNA that is present only at the anterior can actually be translated.
Hunchback protein was examined in embryos that lacked nanos activity. The hunchback mRNA is translated through the entire embryo in embryos that lack nanos activity. This means that when you remove nano, somehow the translated regulation that would normally be on the hunchback mRNA so that it does not get regulated in the middle, but only at the presumptive anterior is suddenly relieved and it gets translated everywhere.
Now we know that nanos protein forms a nice gradient that interacts with the hunchback mRNA at specific sites in the 3’ UTR so that those mRNAs cannot be translated. It is only in the regions where there are no nanos protein that the hunchback mRNA will be translated into protein which gives rise to the important red crescent that will specify the head and anterior structures of the fly. This is an example of translation regulation in a growing animal.
Regulation of the stability of ferretin mRNA.
- FERRITIN is an intracellular protein that binds iron ions, thereby preventing the accumulation of toxic levels of free iron ions.
- Ferritin is a very important protein to maintain intracellular levels of iron.
- If the iron levels get too high, ferritin will act like a sponge to sequester the iron such that the levels can be maintained within the cell at a concentration that is reasonable for the viability of the cell.
During high iron conditions, you want ferritin to be expressed at reasonable levels within the cell. In high iron conditions, the IRE-BP is in a non active concentration and it will never interact with its targets that are present at the 5’ end of the untranslated region of the ferritin mRNA.
Although there are stem loops that are present around the IREs, they don’t block the scanning complex as it makes its way through the 5’ UTR looking for the UG cpfpm. As a result, you make lots of ferritin protein that can sequester the intracellular iron so that it does not cause not kill the animals.
When the concentrations of iron drop, the last thing you want is to sequester your intracellular iron and ferritin. Luckily, in these situations, the IRE binding protein IRE-BP takes on an active conformation and in doings so it will interact with those IREs that are present on the stem loops and the 5’ untranslated region of the ferritin mRNA. Through this interaction, that is so tight, the scanning complex is blocked. The interaction is so tight that the scanning complex cannot kick it off. Therefore, it blocks the formation of the ferritin protein (no translation of the mRNA can take place).
This is all based on IRE-BP interacting with the IREs present in the stem loops and the 5’ untranslated region of the ferritin mRNA. This is translational regulation through an interaction between RNA binding protein and these particular ion response elements and its target.
Developmental timing mutants in C. elegans
Developmental timing mutants in C. elegans helped to elucidate a new pathway.
* Developmental timing — They were working on understating how C elegans genes were responsible for a number of major changes that allowed animals to more or less grow up. They go through 4 larval stages (L1, L2, L3,L4).
Studying one particular mutant Lin-4: lineage abnormal 4. They constantly reiterate the L1 specific event over and over again.
* 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 then it has to be processed, after which it binds its target sites and affects translation of the lin-14 mRNA.
* Lin 14 is required to specify the first larval stage but then its levels have to go down in order to transition into the second larval stage. The lin 4 microRNA binds to the Lin 14 which destabilizes the mRNA and represses its translation. This allows to move to the next larval stage.
One of the gene products he was working on repeated the L1 stage over and over, just like Lin 4, suggesting that there is an interaction . When he injected this DNA in the C elegance it could correct the mutant defect. He found the gene - there was no protein in the gene (DNA sequence that does not encode any protein, no open reading frame). He found that what was rescuing the animals was the fact that this segment made a small RNA that was about 62 nucleotides in length and that small RNAs called Lin 4 and when animals were mutant they did not express or couldn’t make that small RNA anymore. They could not get out of L1. He notices that that particular variant of Lin 14 mRNA didn’t have a 3’ UTR and that is why those animals were reiterating the L1 stage.
Now we know that more than 60% of our genes are regulated by these little RNAs that started out by guys trying to figure out how timing took place in a C elegan
