RR13: post transcriptional regulation

Question 1

what else does the scanning complex do as it advances along the mRNA?

Answer 1

it is also involved in knocking other things off the mRNA (nuclear proteins, proteins that are not supposed to be there)
Quality control of cytoplasmic remodeling: nuclear proteins are removed by RNA helicase, but that is not actually sufficient, cells are not perfect, sometimes nuclear proteins get through that mechanism
that is why the first round of translation is different from the others

Question 2

what do errors in the mRNA give rise to?

Answer 2

an in frame stop

Question 3

what does an in frame stop lead to?

Answer 3

if the in frame stop is translated it will give rise to truncated proteins

Question 4

what do truncated proteins do?

Answer 4

70% of the time they are not an issue, but depending on the nature of the protein they can cause damage

Question 5

explain how a steroid hormone receptor truncated protein could cause damage?

Answer 5

• Modular: ligand binding domain and transcriptional activation domain
• In frame stop permits the truncated protein to bind to its steroid hormone (estrogen) and it can still bind to DNA but it doesn’t have a transcriptional activation domain
• Under those circumstances, that protein can sit down on all the DNA binding sites that it recognizes in estrogen bound form but it can’t do anything: competing for active positions with the real receptor that is correctly built
• Can give rise to loss of function phenotype if you rely on those genes that should be activated
• dominant negative effect: not a full on mutation but it damages the homeostasis of the cell
• same is true for other receptors that require modules
• You can bind the ligand but not carry out the function: bind up the ligand but nothing is working
• Recognized as poisonous by the cell: you dont want those proteins around because they interfere with normal cellular functions that need the full protein

Question 6

what happens when a ribosome hits an in frame stop and dissociates?

Answer 6

any proteins that are still on the mRNA after that part will not be removed, it will be untranslated also

Question 7

what happens when the cell recognises those in frame stop abnormalities?

Answer 7

• Sends off alarms within the cell and sets of a process called NMD: nonsense mediated decay
• Brings in devoted exoribonucleases to eliminate that specific mRNA, important to maintain homeostasis
  very efficient quality control mechanism

Question 8

why are cell generation times and mRNA half lives longer for mammalian cells?

Answer 8

we maintain homeostasis better, better temperature control, constant flow of nutrients

Question 9

why do some mRNAs have to be purposefully destabilised? and give examples and consequences

Answer 9

• Some mRNAs have to be purposefully destabilized, only supposed to be there very shortly, such as mRNAs linked to the cell cycle
• Very important during development or growth, but once the tissues differentiate, you want to make sure that all those cell cycle mRNAs are destabilized
• Continuous cell cycle = tumor formation
• Cytokines or mRNAs involved in immune responses that are radical and dramatic: you want them to do the job very quickly but not stick around for too long

Question 10

in what manner is mRNA degradation regulated?

Answer 10

very tightly, needed to maintain those steady state levels
transcription has to switch depending on the environment

Question 11

what is an example of a sequence that is involved in mNRA regulation?

Answer 11

• sequences in the mRNA itself is involved in this regulation
• GMCSF is important for driving the proliferation of a number of immune cells
• There is a sequence in the mRNA of that GMCSF that déstabilises it, because you dont want it around for too long or it will cause problems
• Too many white blood cells- leukemia or other diseases
• Within the 3’ UTR of that mRNA there are some elements that are rich in AUUUA sequences

Question 12

how can the efficiency of those mRNA destabilising sequences be tested?

Answer 12

• If you introduce that sequence in the 3’ UTR region of the beta globin in gene from the GMCSF mRNA, you change the half life from 10 hours to 1-2 hours.
• Control: add to that same region another sequence that isn’t AUUUA to show that it’s that specific sequence
• That variant other sequence doesn’t have an effect on the hald life of the variant mRNAwh

Question 13

what does this sequence do? how does it work?

Answer 13

• Recognized by critical proteins that will recruit an assembly of exoribonucleases that will destroy the mRNA

Question 14

what are some enzymes involved in mRNA degradation and in what direction do they work?

Answer 14

deadenylase complex: deadenylates the poly A tail in the 3’-5’ direction
exosome: RNA degradation machine that is recruited to chew up RNA from 3’ to 5’
the 5’ end is attacked by enzymes that will remove the 7’ methyl guanosine cap (decapping enzymes) which exposes that 5’ end to be degraded by XRN1, which works in 5’ 3’ direction

Question 15

where does decapping and degradation in 5
3’ direction take place?

Answer 15

takes place in the p bodies, liquid liquid condensates, membraneless organelles which bring together those enzymes responsible for destabilizing the mRNAs

Question 16

how does the exosome work?

Answer 16

• It will take in the RNA and will suck it up into a tube, and at the very end of that tube there are two different ribonucleases: exoribonucleases, and if that one perchance misses a fragment, there is an endoribonuclease right next to it that will take care of it: very efficient
• Takes place in the P bodies

Question 17

what happens if an mRNA is cut in the middle by an endonuclease?

Answer 17

• if an mRNA is cut in the middle by an endonuclease, that is the kiss of death, it will destroy it very rapidly
• Activate degradation on both sides
• Important for RNA mediated interference

Question 18

how are intracellular iron concentrations maintained by mRNA destabilising?

Answer 18

• Transferrin receptors (TfR) is required to get iron into the cells when the cell needs more iron
• The TfR has important secondary structures in the 3’ UTR which contain specific elements called iron response elements (IRE)
• Under normal conditions, TfR will bring in iron if the cell needs, but if the levels of iron increase, these stem loops in the 3’ UTR that have associated IRE have AU rich elements that will recruit proteins to destabilize the mRNA, you dont want more iron you dont need TfR
• MRNA is rapidly degraded and you dont bring any more iron into the cell
• IRE-binding protein is an RNA binding protein that interacts with IREs
• IRE-BP isn’t in its active conformation when the iron levels are high, but when iron levels drop, it takes on an active conformation, and it will then interact with the IRE sequences, it protects the stem loops from interaction with the proteins that normally interact with AU elements to déstabilisé the mRNA, so stabilises the mRNA, allows it to be translated at a higher rate, gives rise to TfR which brings in more iron into the cell
• All based on the conformation of IRE-BP so you only make the protein required for this function when it is needed

Question 19

how is iron involved in cells?

Answer 19

needed for a number of enzymatic reactions, but extremely toxic, so levels have to be tightly regulated

Question 20

what is another way to regulate gene expression that is not destabilising the mRNA? and how is that shown?

Answer 20

• you can also regulate gene expression by blocking/affection translation of those mRNAs
• it is not the stability of the mRNA that is the case, but the way in which its translated: translational regulation
• levels of an mRNA do not change in a cell (northern blot)
• when you compare it to a western blot, over time the protein levels diminish
• Not dependent on the mRNA levels, but how those mRNAs are being read, post transcriptional

Question 21

how is translational control shown in drosophila embryos?

Answer 21

• interaction between two important proteins and mRNAs and their roles in specifying the posterior and anterior segments of the animal
• hunchback is a transcription factor that’s involved in specifying structures that will give rise to the front part of the animal: anterior specific factor
• Nanos proteins: specify the posterior end of the embryo
• If we examine the mRNAs in a very early embryo, the hunchback mRNA is all through the embryo, there is no specific segment associated with hunchback mRNA
• Nanos is largely enriched at the posterior
• If we look at the proteins that correspond to those mRNAs, the hunchback protein is highly enriched at the anterior portion, forms a steep gradient so that the levels diminish towards the middle
• The opposite is true for the nanos protein (lots at the posterior end, gradient and very low at the middle)
• If you remove the Nanos gene in the early embryo, there’s no change to hunchback mRNA (still everywhere), but the protein is also expressed everywhere: result is lethal, where the embryo has anterior but no posterior structures
• Suggests that mRNA for hunchback is not being translated in the posterior sector because nanos is there, but when nanos is removed it can be translated everywhere
• Nanos is an RNA binding protein, a zinc finger that interacts with sequences in 3’ UTR of hunchback mRNA, which will make it not properly translated
• The RNA is there, not eliminated but blocked from being translated
• Translational regulation

Question 22

how can iron concentration be regulated by a mechanism that is not mRNA destabilisation?

Answer 22

• Ferritin: critical protein for iron homeostasis
• Sequesters intracellular iron so that it can’t participate in reactions if the levels are too high, allows for normal cell activities without poisonous effect of high iron concentrations
• in high iron concentrations, you want ferritin to be there
• On ferritin mRNA there are two stem loops in the 5’ UTR that also have IREs
• When high iron, the IREs are not bound by IRE-BP (which is only active when low iron)
• The scanning complex can get through those stem loops and give rise to those proteins
• If iron drops, IREBP becomes active, interacts with the IREs on the stem loops, tightly binds, and blocks the advance of the scanning complex so that ferritin cannot be produced, and iron can be used by the cell to carry out reactions
• Once again all depends on IREBP

Question 23

how does C.elegans grow?

Answer 23

• C.elegans goes through a number of larval stages: L1,2,3,4 and then mold into an adult worm
• Look all similar except they get bigger

Question 24

what does the lin4 mutant do?

Answer 24

goes through L1 but then repeats that first larval stage over and over again, never grows up

Question 25

which allele is responsible for specifying L1?

Answer 25

lin14

Question 26

what is the characteristic of the mutation on lin14 that had the peter pan_

Answer 26

this mutation lacked the 3’ UTR

Question 27

what needs to happen to lin14 for L2 to happen?

Answer 27

lin14 has to disappear

Question 28

what is the nature of lin4 and what does it do?

Answer 28

• chopped it down to a region where they knew that lin4 had to be there, but there was no protein coding gene, there was no ORF
• Lin4 is a small non coding RNA that binds to sequences in the 3’UTR of lin 14 which blocks the expression of lin14 at the translational level, but also destabilizes mRNA

Question 29

what kind of gene is affected by the lin4 mutant?

Answer 29

narrowed to a small chunk of DNA, small section that gives to a small RNA –> 64nucleotides precursor that gets processed to a 21 nucleotide RNA

Question 30

how does lin4 create lin14’s demise?

Answer 30

small lin4 RNA has limited homology to the 3’ UTR of the lin14 mRNA, suggesting that lin4 is acting in an antisense manner to bind to those sequences in the 3’ UTR

Question 31

what happens when the 3’ UTR is missing in lin14?

Answer 31

that regulation with lin4 cannot take place, lin14 sticks around and the L1 stage is repeated

Question 32

what are the two ways in which small RNAs can affect mRNA/gene expression?

Answer 32

affect the direct translatability of that mRNA or induce the deadenylation of that mRNA and eventually the loss of that transcript

Question 33

what protein transcribed miRNAs? and where?

Answer 33

pol II in the nucleus

Question 34

how are miRNAs made/processed and how do they work? (the whole process)

Answer 34

• they start out as big pol II transcripts and get processed down to pre-mRNA, then leave the nucleus with exportin 5 and then once it gets to cytoplasm
• interacts with an RNAase III like enzyme called dicer
• Dicer will recognize the double stranded miRNA precursor and then chop it into specific small double stranded entities: miRNAs
• dicers will hand off this miRNA (double stranded) to another complex called the RISC complex: RNA induced silencing complex
• at the core of this complex is an argonaute protein: highly conserved proteins because they have an ATP helicase activity associated with them that allows them to separate the two strands of the double stranded RNA and use one of them as a guide with which it will accompany to its final destination: mRNA targets that have limited antisense homology to that guide miRNA
• the RISC complex with the miRNA will carry out one of those two events: blocking translation or summoning in the deadenylation complex to déstabilise the target mRNAs
• Target mRNAs can constitute entire families of gene products based on the presence of these sequences in the 3’ UTRs
• Through that interaction in an antisense manner you can block gene expression

Question 35

what happens once you stop producing that miRNA?

Answer 35

not finite, all the mRNAs will be expressed again

Question 36

what is the whole process referred to as?

Answer 36

developmental fine tuning

Question 37

what processes are miRNAs involved in?

Answer 37

metabolism, tissue growth, neural development, developmental timing, stem cell biology, maintaining pluripotency and also in cancer

Question 38

what percentage of our coding genes are under some form of miRNA regulation?

Answer 38

60%