W11L2 translational regulation by uORFs in the eukaryotes

Question 1

The probability of an ORF at a given length

Answer 1

The probability of an given length is : 1/64 x (61/64)^n-2 x 3/64
The chance of having a start codon
The chance of not being a stop codon
The chance of stop codon

Question 2

Expected and actual ORF length

Answer 2

• the chance of finding a short reading frame should be high and decrease as the ORF lenght
  -This is not what was observe, rise and then plateau in the peptide length
  -due to selection of functional protein

Question 3

The problem of annotating a genome

Answer 3

• the algorithm can ignore short ORF even if it is fuctional

Question 4

Small ORF location and fuction

Answer 4

-small ORF can be identified throughout eukaryotic genome
-uORFs play a role in translational regulation

Question 5

translational control of amino acid starvation

Answer 5

Gcn2 is a repressor of translation during aa starvation
Gcn4 is a transcriptional activator of amino acid biosynthesis genes
GCN4 is translationally upregulated via Gcn2

Question 6

Gcn2 fuction

Answer 6

-Gcn2 is a eIFal kinase
-phosporolisation of eIF2, which cause it to inhibit translation
-globally repress translation initiation by limiting availability of charged initiator tRNA

Question 7

How is GCN4 regulated

Answer 7

-GCN4 is not transcriptionally regulated by AA starvation, but its translation is strongly increased
-GCN4 has a long 5’UTR containing 4 uORFs
- in normal condition, ribosome initiatiate translation at the 1st uORF and continue scanning , and disassociate at uORF this occur and there is very low translation of the GCN4
- in lack of aa condition, skip uORF and translate GCN4
-Inhibition of eIF2 by Gcn2 kinase reduces loading of tRNAMeti

Question 8

Mutation of uORF in GCN4 to study fuction

Answer 8

Mutation of all uORFs abolishes translational repression
But mutation of uORF1 alone reduces expression >50% (non-indictable)
-lead to constitutive phenotype
-only uORF1 and 3 or 4 to have a wildtype phenotype

Question 9

Key features of GCN4 5’UTR

Answer 9

Continued scanning of uORF1 (~50%) following translation
Distance between uORF1 and uORF4 Affect GCN4 translation rate (longer = less likely)

Question 10

ATF4 in animals is analogous to GCN4 in yeast

Answer 10

-ATF4 is a transcriptional activator under AA starvation
-Like GCN4, ATF4 levels increase by eIF2⍺ phosphorylation

Question 11

How to detect uORF

Answer 11

-Using ribosome profiling to detect uORF
-leaky scanning will allow for multiple initiation site

Question 12

Using ribosome profiling to detect uORFs in GCN4

Answer 12

-There is an additional non-AUG ORF upstream of ORF1
-143 non-AUG ORFs translated in S. cerevisiae (~20% of footprints in 5’UTRs)

Question 13

How wide spread are uORF in eukaryote genome

Answer 13

-50% of vertebrate mRNAs contain uORF or oORFs

Question 14

Conservation of uORF

Answer 14

-uORF are mostly not conserved at amino acid sequence
-For most uORF, peptide sequences is likely not important

Question 15

Characteristic of uORF

Answer 15

-they are too short
-they don’t all start with AUG
-they are not conserved
-UTRs are not UTRs

Question 16

What is the global effect of having uORFs in 5’UTRs

Answer 16

Increasing uORFs in mRNAs reduces translational efficiency of mORF

Question 17

How uORFs and oORFs mediate translational repression experiment

Answer 17

-Increasing number of uORFs enhances translational repression (remember, leaky scanning)
-Weak Kozak sequence prevents translational repression

Question 18

factors that alter efficiency of uORF-mediated translational repression

Answer 18

-Distance to the cap (more likely if long)
-uAUG context
-length (longer=more repressive)
-secondary structure
-conservation
-number
-position of stop codon
-length of the intercistronic region

Question 19

eukaryotic riboswitch controls uORF translation

Answer 19

-In eukaryotes, riboswitches can control alternative splicing
-Thiamine pyrophosphate riboswitch controls NMT1, a gene required for thiamine biosynthesis
-TPP riboswitch and 2x uORFs in 5’UTR NMT1
Binding of TPP prevents splicing to remove uORFs -> translational repression of NMT1 ORF