Cytoplasmic mechanisms for post-transcriptional control

Question 1

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.

Answer 1

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.

Question 2

What is the exon junction complex made of?

Answer 2

Core complex that includes EIF4AIII and Magoh

Question 3

What is the role of Magoh?

Answer 3

Locks eIF4III in its RNA-bound conformation

Question 4

What are the four functions of the exon junction complex?

Answer 4

1. Localization
2. Translation enhancement through ribosome interaction
3. Nonsense mediated decay
4. RNA export

Question 5

How can we test that the EJC plays a role in localization?

Answer 5

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.

Question 6

What are the steps of nonsense mediated decay?

Answer 6

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

Question 7

What is the role of nonsense mediated decay?

Answer 7

mRNAs with stop codons before the last splice junction are still exported from the nucleus to the cytoplasm. NMD targets them for degradation.

Question 8

Give an example of how cytoplasmic polyadenylation helps regulate translation initation.

Answer 8

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.

Question 9

Give a concrete example of local translational regulation.

Answer 9

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

Question 10

Give other examples where IREs are used for translation control.

Answer 10

In picornaviruses, cell cycle, programmed cell death

Question 11

Explain the TOR pathway when ATP levels are high and there are sufficient levels of nutrients.

Answer 11

1. RagA is in its activated GTP-bound state
2. TCS1 and TCS2 are sensors and have GAP activity for Rheb
3. At high ATP, TCS1 and TCS2 are inactive, so Rheb is in its active GTP-bound form
4. mTOR is activated
5. mTOR hyperphosphorylated 4E-BP, leading 4E-BP to release eIF4E which associate with capped mRNA and eIF4G, resulting in translation initation

Question 12

Explain the TOR pathway when there are low levels of nutrients.

Answer 12

1. Low energy means an increase in cAMP
2. cAMP activates AMPK which phosphorylates TCS2 at the GAP activity site
3. TCS2 converts Rheb to inactive GDP-bound state
4. TOR is inactive
5. 4E-BP is hypophosphorylated, which increases the affinity between 4E-BP and eIF4E, resulting in translation inhibition

Question 13

What does rapamycin do in the TOR pathway?

Answer 13

It inhibits TOR, leading to suppression of cellular processes

Question 14

What does the unfolded protein reponse address?

Answer 14

The UPR is a cellular stress response mechanism that is activated when there is an accumulation of unfolded or misfolded proteins in the ER.

Question 15

What is the long-term solution of UPR?

Answer 15

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

Question 16

What is the outcome of UPR?

Answer 16

Translated Hac1 transcription factor which moves in the nuclus and activates transcription of genes encoding several protein-folding catalysts

Question 17

What is the short-term solution to the protein folding problem?

Answer 17

Short-term solution => Downregulated translation
* BiP binds to unfolded proteins, making it unable to bind to PERK or IRE1 protein
* When BiP is absent, PERK dimerizes and phosphorylates eIF2-GDP, which tightly binds to GEFand prevents GEF from exchanging GDP for GTP on eIF2, rendering all eIF2 inactive, causing a downregulation of translation

Question 18

What happens when IRE1 or PERK activity is impaired?

Answer 18

Protein folding in the ER cannot be moderated. Folding capacity of ER is overwhelmed and this perturbs organelle function.

Question 19

How is Lin-14, Lin-4, and Lin-28 related to each other?

Answer 19

1) lin-4 which represses lin-14 and lin-28
2) lin-14 which specifies early events
3) lin-28 which specifies late events

Question 20

Explain the reverse genetics experiment that led to the discovery of RNA interference.

Answer 20

• Inject C.elegans with 3 variants of unc-22 RNA: 1) sense RNA, 2) Antisense RNA, and 3) Double-stranded RNA
• Examine the resulting phenotypes
• Controls for further experimentation
• Use 10-20 dsRNAs and test whether target gene is downregulated using qRT-PCR
• Test for specificity by injecting dsRNA, and then performing a rescue experiment
• Compare introns and exons against dsRNA => exons lead to mutated phenotype whereas introns don’t, suggesting that the mechanism takes place in the cytoplasm after intronic segments have been spliced out
• Stoichiometry: varying the ratio of dsRNA to mRNA to evaluate enzymatic activity
• RESULTS:
• Sense RNA -> Wild-type phenotype
• Antisense RNA -> Wild-type phenotype
• dsRNA -> Twitcher as unc-22 activity is decreased

Question 21

What is the mechanism of RNA interference?

Answer 21

• Initially, miRNAs are transcribed as longer RNA molecules (pri-miRNAs which may contain hairpin structures)
• Pri-mRNA undergoes processing in the nucleus by Drosha-Pasha complex, which cleaves it to produce a miRNA precursor which typically has a stem-loop structure
• miRNA precursor is then exported into the cytoplasm
• miRNA precursor or dsRNA is cleaved by Dicer enzyme into double-stranded short interfering RNA (siRNA) or miRNA that contain 21-23 nucleotides hybridized to each other such that the 2 bases at each 3’ end of each strand are single stranded (overhangs)
• siRNA or mature miRNA are assembled into a RISC complex in which the short RNAs are bound by an Argaunaute protein
• RISC complex induces target mRNA cleavage
• In cases of perfect base-pairing between the RNA and RISC, causes siRNA interference and degradation is induced
• In cases of partial complementarity, miRNA interference causes downregulation of translation is induced

Question 22

What happens when there is perfect base-pairing between the RNA and RISC?

Answer 22

In cases of perfect base-pairing between the RNA and RISC, causes siRNA interference and degradation is induced

Question 23

What does the Drosha-Pasha complex do?

Answer 23

Pri-mRNA undergoes processing in the nucleus by Drosha-Pasha complex, which cleaves it to produce a miRNA precursor which typically has a stem-loop structure

Question 24

What does the Dicer enzyme in the RNA interference pathway do?

Answer 24

miRNA precursor or dsRNA is cleaved by Dicer enzyme into double-stranded short interfering RNA (siRNA) or miRNA that contain 21-23 nucleotides hybridized to each other such that the 2 bases at each 3’ end of each strand are single stranded (overhangs)

Question 25

What happens in cases of partial complementarity between RNA and RISC?

Answer 25

In cases of partial complementarity, miRNA interference causes downregulation of translation is induced

Question 26

Give a concrete biological model where RNA interference is important.

Answer 26

Turnip crinkle virus.
* dsRNA virus enters host plant
* Host has DCL dicer that processes viral dsRNA into siRNAs that are targeted by RISC, and then degraded
* To counteract this, viruses produces a viral suppressor protein that represses DCL4, blocking the degradation of viral RNA

Question 27

Give an example for which miRNAs play an important role.

Answer 27

Measure maternal mRNA and miRNA abundance in function of time, through development
* Dicer -/- => maternal RNA persists after mid-blastula transition
* miR430 -/- => maternal RNA persists after mid-blastula transition
* RESULT: After fertilization until the mid-blastula transition, miRNA430 abundance is low while maternal RNA abundance is high. Once zygotic transcription begins, miR430 expression is activated and its level rise

Question 28

What is the role of cytoplasmic polyadenylation?

Answer 28

Helps regulate translation initation.