L33 Translational control in Eukaryotes by uORFs

Question 1

How long is an ORF?

Answer 1

see onenote slides

100nt is conventionally thought as a functional ORF

Question 2

Translational control by uORFs

Answer 2

see onenote

small ORFs identified throughout eukaryotic genomes

Question 3

Translational control of aa starvation - Gcn2, GCN4

Answer 3

see onenote slides

GCN4 = transcriptional activator of aa biosynthesis genes

GCN2

• Global regulator of translation
• Under aa starvation, it globally represses translation
• It activates GCN4 through uORFs

Question 4

GCN2

Answer 4

see onenote

is a e1F2alpha kinase

globally represses translation initiation by limiting availability of charged tRNA

GCN2

• Activated by aa starvation
• Repressed by aa presence

Question 5

GCN4

Answer 5

see onenote slides

not transcriptionally regulated by aa starvation but its translation is strongly increased

• has a long 5’ UTR containing 4 uORFs

Question 6

GCN4 - non-starvation

Answer 6

see onenote

non-starvation => low Gcn4

Question 7

GCn4 - starvation

Answer 7

see onenote

starvation => increased Gcn4 translation

inhibition of e1F2 by Gcn2 kinase reduced loading of tRNA-met-ini => small sub-unit scans through uORFs => translates GCN4

Question 8

Mutation of uORF AUGs to study function

Answer 8

see onenote slides

mutation of all uORFs abolishes translational repression

uORF1 of GCN4 retains small sub-unit of ribosome on mRNA, continues to scan along mRNA, looking for another AUG
=>
If we mutate first uORF, there is low expression of lacZ as uORF1 is the ORF that retains the small subunit of ribosome. Without functional uORF1, the ribosome would disassociate and translation cannot occur

You only need uORF1 and a second uORF down stream for WT phenotype

Question 9

Key features of GCN4 5’UTR

Answer 9

see onenote

Question 10

ATF4 in animals

Answer 10

analogous to GCN4 in yeast

ATF4 a transcriptional activator under aa starvation

like GCN4, levels of ATF4 increase via e1F2-alpha phosphorylation

Question 11

Using ribosome profiling to detect uORFs

Answer 11

see onenote slides

REMEMBER - leaky scanning will allow multiple initiation sites

found non-AUG ORF upstream of ORF1

Question 12

Non-canonical start important for translational regulation

Answer 12

see onenote

uORF regulating translation of vitamin C biosynthesis in plants

mutating non-canonical start (ACG) to AUG abolishes of translation of mORF i.e. leaky scanning required for effective translational regulation

Question 13

uORFs mostly not conserved at aa sequence

Answer 13

orthologous genes contain uORFs in 5’UTR

for most uORFs, peptide sequence likely not important - uORFs are mostly just regulatory DNA, not protein coding elements

Question 14

uORFs break the rules

Answer 14

see onenote

Question 15

uORFs mediate global translational repression

Answer 15

increasing uORFs in mRNAs reduce translational efficient of mORF

effect of uORF in 5’UTR greater/equal to miRNA site in 3’UTr

Question 16

uORFs and oORFs mediate translational repression

Answer 16

see onenote

increasing number of uORFs enhances translational repression (remember leaking scanning) hence,
weak kozak sequence prevents translational repression

Question 17

All uORFs are not created equal

Answer 17

see onenote

many factors alter efficiency of uORF-mediated translational repression

Question 18

Eukaryotic riboswitch controls uORF translation in N.crassa

Answer 18

see onenote slides

riboswitches control alternative splicing in eukaryotes

Thiamine pyrophosphate (TPP) riboswitch controls NMT1, gene required for thiamine biosynthesis

Binding of TPP prevents splicing to remove uORFs => translational repression of NMT1 ORF