L32 Regulation of translation in eukaryotes

Question 1

Eukaryotes vs Prokaryotes

Answer 1

Eukaryotes:

• no RBS
• mRNA recognised by 5’ cap
• mostly monocistronic
• polyA tail

Question 2

5’ cap

Answer 2

see onenote

Question 3

Initiation of translation in eukaryotes

Answer 3

see onenote slides

1. cap recognition
2. scanning by small subunit - kozak
3. associaiton of large subunit
4. elongation

Question 4

Leaky scanning

Answer 4

see onenote

can produce alternative proteins

Question 5

Features of translation in eukaryotes

Answer 5

• physical separation of transcription and translation
• interaction between 5’ cap and polyA tail

5’ cap and 3’ poly A tail interact to form a loop

• Reduce translational efficiency if poly A tail is shortened this is due to the reduction of proximity
• If the association is tight, when the mRNA falls off from the ribosome at the 3’ end, it can be reinitiated at the 5’ more efficiently and quickly as the 5’ end is nearby

Question 6

IRES

Answer 6

see onenote

cap-independent translation initiation in eukaryotes

very rare

viruses use IRES for translation in host cells

Question 7

Detecting translatome

Answer 7

see onenote

Question 8

RNA-seq - what does it tell us?

Answer 8

steady state transcription levels in a cell/tissue

cheap and easy

Question 9

Translatome - ribosome profiling

Answer 9

see onenote

tells us position of ribosomes on mRNA => which ORFs are being activity translated

Question 10

Translation efficiency equation

Answer 10

see onenote

Positive = translated efficiently and vice versa

Question 11

Translational regulation under starvation

Answer 11

see onenote

Question 12

Regulation of mRNA recognition by TOR kinase

Answer 12

see onenote slides

e1F4F complex required for cap-dependent translation

effect of mTOR inhibition on translation requires 4E-BP

Question 13

Global vs specific regulation by mTOR

Answer 13

see onenote slides

99% mRNA translationally regulated by mTOR

BUT…SOME mRNA more susceptible to TOR than others:

• Strong enrichment of mRNA associated with translation are down regulated by mTOR
• mRNA with IRES are CAP independent, don’t required recognition of 5’ cap, aren’t down regulated by mTOR

Question 14

5’ TOP mRNA

Answer 14

5’ TOP mRNAs

• Have 5’ end very rich in C’s and U’s due to association of 4E-BP binding protein to 5’ region of mRNA, better affinity for 4E-BP. If the sequence has higher affinity for 4E binding protein, would be more sensitive to mTOR regulation
• If they have TOP mRNA, strong skew of regulation by mTOR

Question 15

mRNA-specific translational repression by 4E-BP

Answer 15

see onenote

polar nucleolisation of mRNAs in Drosophila oocyte

• During trafficking process, need repression of translation of that mRNA

Question 16

mRNA-specific translational repression by 4E-BP - Oskar

Answer 16

see onenote

posterior localisation of maternal oskar mRNA establishes polarity in Drosophila oocyte

Question 17

How is translation of Oskar mRNA repressed during transport?

Answer 17

see onenote

mRNA-specific translational repression by a cup, 4E-BP

abundance of cup is too low to repress all mRNA, Bruno required to recruit Cup to Oskar mRNA

Question 18

Iron homeostasis - balancing deficiency and excess

Ferritin vs Transferrin

Answer 18

see onenote

Ferritin - iron storage protein: buffers against toxicity and deficiency, less ferritin produced when there is lack of Fe

Transferrin receptor is an Fe uptake protein, required for Fe uptake under low Fe conditions (opposite to ferritin)

Question 19

Aconitase in a cytosolic Fe-containing enzyme

Answer 19

Sufficient iron in cell:
IRP1 = aconitase, Fe containing enzyme

Insufficient:
Fe-free aconitase becomes an RNA binding protein

• Aconitase has dual function
• IRP1 acts as aconitase, binds to iron when there’s too much iron
• IRP1 becomes an RNA binding protein when there isn’t enough iron. Binds to IRE (iron regulatory element).

Question 20

Regulation of translation initiation by IREs - starvation vs excess

Answer 20

see onenote slides

IRE = secondary structure in mRNA

Question 21

Regulation of mRNA stability by IREs

Answer 21

see onenote

IREs present in 3’ UTR of transferrin receptor mRNA

IREs can have opposite effects on post-transcriptional regulation

Question 22

Translational control by miRNAs

Answer 22

see onenote slides

miRNAs contribute to translational repression but mechanism not well defined

RISC complex

• Repress translational initiation, reduce translation efficiency
• Interferes with close-looped structure
• GW182 interacts with polyA binding protein, disrupts close-looped structure => reduced translation efficiency