Control of eukaryotic protein synthesis

What are the advantages of translation control?

One advantage of translational control is the speed of the effect on protein levels. If transcription was not regulated, it would take much longer since introns can be huge. A second advantage is that it allows local control in a specific part of the cell. Controlling things at different levels allows cells to regulate their energy usage, as protein synthesis is an energy-consuming process.

What situations typically affect translation?

Under conditions of stress, translation is often inhibited transiently until stress is resolved. Translation can also be activated allowing faster growth and proliferation upon stimulation by hormones, GFs, mitogens and cytokines - globally during early development or locally during wound healing etc.

How do the 5’ cap and 3’ polyA tail affect translation?

The 5’ cap and 3’ polyA tail synergistically enhance translation of all eukaryotic mRNAs. This was shown through translation of a luciferase gene (amount of light it generates is proportional to the amount of protein made) without either, with both, and with one or the other. It showed that with both, there is around 800x more translation of the gene. This is because the 5’ cap and polyA tails are targeted during mRNA decay.

What is polysome profiling and how does it work?

Polysome profiling can be used to measure global protein synthesis activity. Firstly, treat cells with cycloheximide to freeze all ribosomes where they are on mRNAs, layer the cell extracts onto 10-50% sucrose gradient in a special centrifuge tube (50% at the bottom), then centrifuge to sediment. It separates so that the heavier stuff deposits near the bottom. 

• A trace shows a large 80S peak which includes free ribosomes, or mRNA with only 1 ribosome bound.

• This can be done under different conditions to analyse their effect on protein synthesis. For example, the addition of increasing amounts of H2O2 shows an increasing 80S peak and reduced polysomes peaks indicating a repression of translation initiation - it does this via oxidative stress. This can also be seen using a 35S methionine labelling of new proteins - visualise less incorporation.

What conditions cause phosphorylation of eIF2?

Oxidative stress activates the kinases Fam69C and GCN2 which phosphorylates eIF2 on its alpha subunit at Ser 51. This inhibits protein synthesis - not all stopped but most are, some mRNAs are resistant to control or have translation activated by different mechanisms to help cells respond to the stress. 

There are several other kinases which all phosphorylate eIF2 but respond to different conditions;

• MARK2 is the kinase activated via cytoplasmic protein misfolding, whereas PERK is activated by ER-associated misfolding.

• GCN2 is activated by amino acid starvation.

• PKR is activated by viral infection.

• HRI is activated by haem deficiency and heat shock.

How does phosphorylation of eIF2 inhibit translation?

eIF2B is the guanine nucleotide exchange factor for eIF2, promoting eIF2-GTP-met-tRNA TC formation. eIF5 is the GTPase activating protein (GAP) that switches eIF2 at AUG codons. When eIF2 is phosphorylated, it blocks EIF2B GEF activity.

How does Down Syndrome link to eIF2?

Down syndrome occurs when a child has an extra copy of chromosome 21 in each cells’ genome (trisomy 21). This causes physical and developmental delays and intellectual disabilities. It has been linked to this control pathway.

• A mouse model with DS was used, called Ts65Dn. Its 80S peak was higher and its polysomal peaks were lower than normal. It has aberrantly high eIF2alpha phosphorylation by PKR kinase. Reduced protein synthesis affects memory. 

• This means that a transient stress response is good but a prolonged response is bad due to too little translation.

How do 4E-BPs inhibit translation?

eIF4E binds to the 5’ M7G cap structure of mRNAs. It interacts with eIF4G. It also binds to eIF4A, and collectively they form eIF4F which recruits the ribosome and protein synthesis occurs. This step is regulated by eIF4E binding proteins(4E-BPs) which inhibit translation by preventing the interaction between eIF4E and eIF4G as they bind to the same place as the latter protein. This blocks mRNA recruitment and shows down translation. 

• The eIF4G and 4E-Bps have a common sequence motif; YXXXXLΦ. X is any amino acid, and Φ is L, F or M. This sequence binds to the same part of eIF4E.

How does mTOR affect 4E-BPs?

The competition between these two molecules can be controlled by mTOR (mammalian target of rapamycin). mTOR is a protein kinase that receives signals from hormones, GFs etc, and phosphorylates the 4E-BPs which inactivates them. Protein synthesis can occur. 

• 5’TOP mRNAs are most impacted by the mTOR control. They have a 5' terminal oligo-pyrimidine run - at the 5’ end there are runs of Cs or Us (4-17). These are found in ribosomal protein and translation factor mRNAs, meaning they control the translation of everything else.

What is the structure of 4E-BP and how does it change due to phosphorylation?

Unphosphorylated 4E-BP is unstructured except for one alpha helix at the common sequence motif. It binds to eIF4E like a necklace. Phosphorylation of Thr45 and 36 causes a dramatic folding of the central region which changes its common sequence motif into a beta strand. This buries the key Tyr53 (Y) so lowers the affinity for eIF4E >10000 fold, freeing the protein for translation. Other phosphorylation sites include Thr69, Ser64 and 82.

What is the importance of mRNA specific controls?

mRNA specific controls work with the global controls to enable precise regulation of translation of specific proteins according to need - they can escape global regulation. The 5’ and 3’ UTRs are critical for determining variations in translation and the control of translation.

How does the ferritin mRNA control translation?

Ferritin mRNA offers translational control in response to Fe levels in mammals. Aconitase/iron regulatory protein 1 (IRP1) regulates ferritin mRNA translation. Aconitase is a 4Fe-S cluster binding enzyme that converts citrate into isocitrate in the TCA cycle when there is a high concentration of iron. In low concentrations, loss of the Fe-S inactivates the enzyme and the protein transforms into a regulator of specific mRNAs called IRP1. This protein binds a stem-loop structure called IRE in ferritin mRNA 5’ UTR to regulate protein synthesis. Therefore, in high iron, RNA helicase allows 40S ribosomes to scan through an open reading frame (ORF) AUG. Low iron binding of the IRE prevents 40S access and scanning.

• Ferritin binds and stores iron, and acts as a buffer for iron deficiency and iron overload.

How does AAP control translation?

The Arginine Attenuator Peptide (AAP) is a translational controlling uORF. Arginine biosynthesis is translationally regulated by both arginine levels and a uORF encoding AAP (25 amino acids long). When there is lots of Arg in the environment, the fungus (Neurospora crassa) does not need to synthesise it via Arg-2 and vice versa.

• In low Arg levels, ribosomes partially bypass the AAP uORF due to its weak start codon (leaky scanning). ~50% of ribosomes translate AAP, then dissociate at the STOP codon. The remaining ribosomes bypass AAP and successfully translate Arg-2, allowing arginine synthesis. 

• In high Arg levels, excess arginine binds to ribosomes translating AAP. This interaction causes ribosome stalling at the AAP STOP codon, preventing peptide release. Stalled ribosomes block other ribosomes, stopping translation of Arg-2 and halting arginine synthesis.

How does nanos mRNA control translation?

To develop from a single cell to a multicellular organism, one early decision set up an anterior-posterior axis. This requires post-transcriptional control of maternal (egg) derived mRNAs - here mutations in genes controlling normal AP axis development results in defective fly embryos (nanos mutant = no abdomen, bicoid mutant = no head/thorax). 

• Staining reveals that nanos mRNA is everywhere but nanos protein localised at the posterior end. Proteins Cup (4E-BP), Glo and Smaug regulates translation and location of nanos protein.

• In the 3’UTR, there is a stem-loop structure which binds Smaug and Glo - which forms a closed loop between Cup and Smaug to shut down the mRNA and prevents eIF4G from coming in (only occurs in delocalised mRNA) so ribosomes cannot scan. This ensures only specific mRNAs are translationally repressed to localise the protein in the cell and establish polarity. 

• Nano itself acts as a translational regulator of other mRNAs. One of its targets is hunchback mRNA - its mRNA is found everywhere but protein is only at the head end. Nanos is part of the repression complex at the posterior end to prevent hunchback from being translated. These controls ensure proper gene expression patterns and embryonic development.

What is programmed ribosome shifting?

During elongation, the ribosome moves codon by codon to make a protein. However, some mRNAs promote ‘programmed ribosomal frameshifting’ where the ribosome can shift forwards +1 or backwards -1. This is common in viruses and some cellular mRNAs.

How does antizyme control translation?

Antizyme (AZ) causes +1 frameshifting and is found widespread in eukaryotes to control polyamine levels in cells.

• AZ mRNA has an ORF with a STOP codon, then the 0 frame (has a START and STOP) and then another ORF (+1 frame) without a START codon. Some ribosomes just translate the 0 frame, but some start to translate the 0 frame but then shift to the +1 frame. This results in short proteins (ORF1) or longer proteins (ORF1-ORF2). This longer protein is the function AZ enzyme. The shift occurs at the stop codon of the 0 frame, where the ribosome moves one base forwards to produce Asp instead of STOP.

• Immediately downstream of the 0 frame STOP codon is a 3’ RNA stimulatory pseudoknot which stalls the ribosome so that it can judder around to slip into the next sequence. There is also a 5’ stimulatory/slippery sequence upstream of the STOP codon.

• Whether or not frameshifting occurs is controlled by spermidine. In high concentrations, it binds to the ribosome to help promote frameshifting here. It is important to control cellular polyamine levels. 

• The AZ levels control ornithine decarboxylase (ODC) enzyme levels which tightly control cellular polyamine levels. ODC converts ornithine to putrescine when polyamine levels are high, which gets converted into spermidine. AZ synthesis will be stimulated by the frameshift, and it binds to ODC to deliver it to the proteasome to be degraded, meaning there is less spermidine synthesis. AZ also inhibits spermidine uptake. Therefore ODC levels and polyamine levels fall so balance is restored.