Cytoplasmic mechanisms for post-transcriptional control Flashcards
You wish to determine which regions of an mRNA are necessary for localization.
A What is one method you could use to do this?
B Describe one molecular mechanism that explains how an
mRNA localizes.
C Describe one concrete example where mRNA localization is
important.
A. Truncate mRNA in different ways and check where it localizes. (e.g. delete 5’ UTR, 3’UTR and different combinations)
B. Microtubules transport mRNAs to synapses.
C. Actin localizes in lamellipodia.
What is the exon junction complex made of?
Core complex that includes EIF4AIII and Magoh
What is the role of Magoh?
Locks eIF4III in its RNA-bound conformation
What are the four functions of the exon junction complex?
- Localization
- Translation enhancement through ribosome interaction
- Nonsense mediated decay
- RNA export
How can we test that the EJC plays a role in localization?
Test in Drosophila system for the formation of an egg cell, using the Oskar gene.
1. Mutate different introns of the Osk gene and observe the localization within the egg cell.
2. See which intron mutations affect the localization.
Conclude that EJC affects localization.
What are the steps of nonsense mediated decay?
1) Abnormal mRNA with a premature termination codon is produced. The EJC left associates with NMD factor UPF3
2) After export to the cytoplasm, NMD factor UPF2 associates with UPF3 bound to the EJC. A ribosome initiates translation and reaches the PTC
3) eRF1 and eRF3 associate with the ribosome, and associate with UPF1
4) Any EJCs associated with mRNA that were not displaced by the terminating ribosome, interact with terminating ribosome through UPF2 and UPF1
5) This binding induces degradation mechanism
What is the role of nonsense mediated decay?
mRNAs with stop codons before the last splice junction are still exported from the nucleus to the cytoplasm. NMD targets them for degradation.
Give an example of how cytoplasmic polyadenylation helps regulate translation initation.
DORMANT - In immature oocytes, mRNAs have short polyA tails and are translationally dormant. CPEB mediates repression of translation through Maskin binding with eIF4E, blocking the binding of eIF4G, necessary for translation.
ACTIVE - Hormone stimulation of oocytes activates a protein kinase that phosphorylates CPEB, releasing Maskin. The PolyA tail is also lengthened. This induces further phosphorylation cascade, allowing eIF4G to bind to eIF4E to start translation.
Give a concrete example of local translational regulation.
Control of intracellular iron (Fe2+) concentration is a method of local translational regulation, and is regulated by IRE-BPs.
Low Fe2+ -> IRE-BP is active and binds a IRE (internal ribosome entry site) at the 5’ of UTR of the ferritin mRNAs, blocking ribosomes and inhibiting translation initiation
High Fe2+ -> IRE-BP is inactive and cannot bind to ferritin mRNA, allowing for active translation
Give other examples where IREs are used for translation control.
In picornaviruses, cell cycle, programmed cell death
Explain the TOR pathway when ATP levels are high and there are sufficient levels of nutrients.
- RagA is in its activated GTP-bound state
- TCS1 and TCS2 are sensors and have GAP activity for Rheb
- At high ATP, TCS1 and TCS2 are inactive, so Rheb is in its active GTP-bound form
- mTOR is activated
- mTOR hyperphosphorylated 4E-BP, leading 4E-BP to release eIF4E which associate with capped mRNA and eIF4G, resulting in translation initation
Explain the TOR pathway when there are low levels of nutrients.
- Low energy means an increase in cAMP
- cAMP activates AMPK which phosphorylates TCS2 at the GAP activity site
- TCS2 converts Rheb to inactive GDP-bound state
- TOR is inactive
- 4E-BP is hypophosphorylated, which increases the affinity between 4E-BP and eIF4E, resulting in translation inhibition
What does rapamycin do in the TOR pathway?
It inhibits TOR, leading to suppression of cellular processes
What does the unfolded protein reponse address?
The UPR is a cellular stress response mechanism that is activated when there is an accumulation of unfolded or misfolded proteins in the ER.
What is the long-term solution of UPR?
Long-term solution => UPR enhances the ER’s protein-folding capacity by upregulating the expression of chaperone proteins, such as BiP, which helps in protein folding
- Accumulating unfolded protein in the ER bind BiP molecules, releasing them from Ire1. Dimerization of Ire1 activates its endonuclease activity
- Unspliced mRNA precursor encoding the transcription factor Hac1 is cleaved by dimeric Ire1, and the two exons are joined to form function Hac1 mRNA
- Hac1 is translated into Hac1 protein, which moves into the nucleus and activates transcription of genes encoding several protein-folding catalysts