W11L1 regulation of translation in eukaryotes

Question 1

DNA sequencing and protein sequencing

Answer 1

DNA sequencing technology
- very fast, very cheap, very effective
Protein sequencing
- (relatively) slow, expensive, challenging
DNA sequencing technology can be used to quantify rates of translation
-polysome sequencing
-ribosome-tagging/affinity purification
-ribosome profiling (Ribo-Seq)

Question 2

Polysome profilling

Answer 2

• extract RNA still bound to ribosome
  -run through a sucrose gradient to separate by density
  -collect RNA

Question 3

Trap/ ribotag

Answer 3

• antibody to the ribosome protein
  -pulling down the ribosome
  -measure RNA

Question 4

Ribosome profiling

Answer 4

-identify sequence where ribosome bound
- using special nuclease digestion to isolate RNA seq where it is not covered by ribosome
-Tells us position of ribosomes on mRNAs
à which ORFs are being actively translated

Question 5

Calculating translational efficiency

Answer 5

Translational efficiency= ribosome footprint density/ relative mRNA abundance

Question 6

Translational regulation under starvation

Answer 6

• many gene translational efficiency and mRNA change does correlate
  -but there are some that have higher levels of translation

Question 7

Method in regulation of translational initiation

Answer 7

Global regulation of mRNAs at 5’cap
Specific regulation of mRNA by 3’UTR
Specific regulation of mRNA by 5’UTR

Question 8

Regulation of mRNA recognition by TOR kinase

Answer 8

-eIF4F complex required for cap-dependent translation
-growth factor activate mTor kinases which phosphorylates 4Ebp
-p4Ebp cannot bind to eIF4E, translational start
-under low nutrient, TOR kinase become inactivated, 4EBP active, prevent translation

Question 9

Global vs specific regulation by mTOR

Answer 9

-99% mRNA are translationally regulated by mTOr
-based on ribosome profiling of mTOR inhibitor (torin 1) treated mouse cells with double knock out
-The effect of mTOR inhibition on translation requires 4E-BPs
BUT, some mRNAs are more susceptible to TOR than others

Question 10

MTOR and IRES

Answer 10

-as IRES doesn’t have a 5’ cap, it is not affected by mTOR

Question 11

Why is there differences to mTOR susceptability

Answer 11

-due to 5’ TOP sequences affect the binding affinity of 4EBP

Question 12

mRNA-specific translational repression by a 4E-BP in drosophila

Answer 12

• polar localization of mRNA in drosophila. Oocyte
  -Posterior localization of maternal Oskar mRNA establishes polarity in Drosopohila oocyte
  -need to repress Oskar during transport

Question 13

Mechanism of repressing Oskar during transport

Answer 13

-mRNA-specific translational repression by a Cup, a 4E-BP
-Abundance of Cup is too low to repress all mRNAs
-Bruno is required to recruit Cup to Oskar mRNA
- Bruno bind to Bruno respond element in the 3’

Question 14

Iron homeostasis – balancing deficiency and excess

Answer 14

-Fe is required for function of 100s enzymes, especially oxygenic metabolism
Fe is highly reactive and is toxic to cells
- Ferritin is an iron-storage protein:buffers against toxicity and deficiency

Question 15

Control of Ferritin

Answer 15

Sufficient iron in cell:
IRP1 = aconitase, an Fe-containing enzyme (Need iron to fuction)
Insufficient iron in cell
Fe-free Aconitase becomes an RNA binding protein

Question 16

Regulation of translation initiation by IREs

Answer 16

Iron Regulatory Element (IRE) is a secondary mRNA structure
PIC cannot unwind mRNA structure when IRE bound by aconitase/IRP
à Translation initiation is inhibited
à Less Ferritin to make Fe available
In the excess of iron
Fe-binding releases aconitase/IRP from IRE
à Translation initiation proceeds
à More Ferritin to store excess iron

Question 17

Transferin in role in iron uptake

Answer 17

Opposite to Ferritin, Transferrin receptor is required for Fe uptake under low Fe conditions
-IREs are present in 3’UTR of transferrin receptor mRNA
when there is excess of iron
Fe-binding releases aconitase/IRP from IRE
à Nuclease cleavage site is exposed
à Transferrin receptor mRNA degraded
When there is iron starvation
Aconitase/IRP binds to IRE under low Fe
à Protects an nuclease cleavage site
à Transferrin receptor mRNA translated

Question 18

Translational control by miRNA

Answer 18

-miRNAs contribute to translational repression, but the mechanism(s) are not well defined
-possible miRNA interfere with the close circle formation of the mRNA
-RISC complex interferes with eIF4G-PABP interaction
Disruption of mRNA closed loop represses translation initiation
miRNA target site in 3’UTR à Reduced translational efficiency in zebra fish