Signalling Pathways in Translation Regulation

Question 1

How is global upregulation effected?

Answer 1

Global regulation is unspecific, upregulating all translation that is currently occurring by targeting the initiation factors for stimulation or repression. This is especially significant given that this is a major rate limiting step in translation.

Question 2

What are 5’ UTRs important?

Answer 2

The regulatory elements are often contained within the 5’UTR of the mRNA, hence why oncogene mRNAs often have giant 2-3kb long UTRs allowing for another layer of tight regulation post-transcription.

Question 3

How can the mRNA sequence effect or be used to effect its regulation?

Answer 3

• Capping/decapping
• 5’UTR length and secondary structure
• IRES structure
• Short ‘uORF’s in front of the main ORF.
• Polyadenylation and removal thereof
• Binding sites for trans-regulatory factors (proteins, miRNA)

Question 4

What are uORFs?

Answer 4

The short upstream open reading frames (uORFs) are caused by AUGs not associated with a Kodak sequence.

Question 5

What is the primary purpose of the Cap and PolyA tail?

Answer 5

The cap and the polyA tail protect the mRNA from being damages, particularly due to their association with the protective proteins Cap Binding Complex and PAB1.

Question 6

What is the main way of initiating mRNA degradation?

Answer 6

Degradation is usually initiated by the shortening of the PolyA tail.

This is not just for regulation, the mRNA must be degraded when it is no longer needed.

Question 7

Why does shortening the PolyA tail lead to degradation?

Answer 7

This can lead to the protective enzymes of the polyA tail being replaces by 3’-5’ exonucleases such as POP2, CCR4 and NOT proteins. It is also the first step in decapping.

Question 8

How does decapping lead to degradation?

Answer 8

The protective cap binding complex is replaced by a complex composed of LSM 1-7 (a kind of helicase), Decapping enzymes 1 and 2 (DCP1/2) and XRN1, a 5’-3’ exonuclease that degrades the RNA.

Question 9

What is proofreading?

Answer 9

This is the simple matter that ternary complexes that do not have the perfect codon will likely dissociate out of the A site without hydrolysing GTP, so that amino acid is not added.
This requires translation to be a reasonable slow process, or the GTP hydrolysis may occur regardless. This is where there is a balance between speed and accuracy in translation.

Question 10

What is Nonsense Mediated mRNA Decay?

Answer 10

When an mRNA has a premature stop codon or is the consequence is an incomplete protein, so the mRNA is targeted for degradation during its translation.

Question 11

What is the mechanism of Nonsense Mediated mRNA Decay?

Answer 11

Premature stop codons (non-sense mutations) are recognised when not all of the exon junction bound proteins have been removed after translation ceases. These complexes are targeted by Upf proteins that activate decapping enzymes, allowing cytosolic exonucleases to degrade the mRNA.

Question 12

What is Non-Stop Mediated mRNA decay?

Answer 12

If a mutation or error in transcription has led to the lack of a stop codon then this leads to the polyA tail being translated into a long chain of lysine residues.

Question 13

What is the mechanism of Non-Stop Mediated mRNA decay?

Answer 13

Lack of a stop codon (non-stop mutation) is noticed by the binding of Ski7 to the polylysine tail, which interacts with the exosome – an RNA degrading protein complex. It also stimulates Lys specific proteases to break down the erroneous peptide.

Question 14

What are miRNAs?

Answer 14

Micro RNAs (miRNAs) inhibit translation or reduce the mRNA level through degradation targeting or by directing the mRNA to processing bodies for storage.

The miRNA often resides within a larger RNA-Induced Silencing Complex (RISC).

Question 15

How are miRNAs produced?

Answer 15

From an ss pri-miRNA transcript that folds to form an imperfectly base paired hairpin loop, the tails on either end of which are cleaved off by an enzyme complex called DGCR8/drosha.

This is translocated to the cytoplasm via exportin5 and the loop end is cleaved off by Dicer/TRBP. The selected strand is then loaded into an Ago protein to form a RISC.

Question 16

What are the effects of miRNAs?

Answer 16

Micro RNAs are thought to prevent translation by binding with the RISC to the 3’ end of the mRNA transcript and curling it around to that the RISC directly interacts with the ribosome(s) and inhibits their activity.

Micro RNAs are also involved in gene regulation at the level of heterochromatin structure, often in an upregulatory manner.

Question 17

How many miRNAs do humans have?

Answer 17

There are thought to be over a thousand miRNAs encoded in our genome that are involved in regulating approximately one third of human genes.

Expression of different miRNAs is often tissue specific or specific to a particular developmental stage.

Question 18

What are siRNAs?

Answer 18

Small Inhibitory RNAs (siRNAs) are RNA duplexes that target mRNA for degradation. They are also known as small interfering RNA and silencing RNA.

These are often artificial or produced by viruses, but can also be endogenous in which case they are bi-directionally transcribed into a long dsRNA that is cut into small strands by a dicer enzyme. The resulting particulates are a perfect antisense for the target mRNA.

Question 19

What is mTOR?

Answer 19

The mammalian Target of Rapamycin (mTOR) is a phosphatidylinositol 3-kinase-related kinase protein. It is an incredibly important regulatory molecule for many processes including ribosomal biogenesis, amino acid transport and initiation of translation.

Question 20

Why is mTOR so important?

Answer 20

It is so important because its activity is controlled by a huge number of variables, allowing it to act as a sensor for the level of various nutrients and metabolites as well as the energy state of the cell.

Question 21

What is Rheb?

Answer 21

Much of the regulation of mTOR goes through Rheb, which stimulated mTOR when GTP bound and inhibits it when GDP bound.`

Question 22

How does mTOR increase the translation rates?

Answer 22

Protein Biosynthesis Upregulation

Ribosome Biogenesis Stimulation

Question 23

How does mTOR upregulate protein biosynthesis upregulation?

Answer 23

mTOR can phosphoactivate S6 Kinase (S6K) which phosphorylates the ribosomal protein S6 and so upregulates the translation process.

mTOR also phosphorylates eIF4E-BP1, lowering its affinity for the initiation factor eIF4E. 4E-BP1 competes with eIF4G for eIF4E but unlike G it has no effect, effectively repressing eIF4E. Hence phosphorylation of 4E-BP1 promotes translation by allowing more eIF4E/G complexes to form.

Question 24

How does mTOR upregulate ribosome biogenesis?

Answer 24

By even further phosphorylation of 4E-BP1 and by an un-elucidated interaction with a protein called UBF, mTOR can stimulate the production of more ribosomes.

4E-BP1 stimulates transcription of 5’TOP mRNA, which encodes a wide variety of proteins involved in translation, both ribosomal proteins and translation factors. UBF stimulation increases transcription of rRNAs.

Question 25

What are the three main ways in which mTOR can be stimulated to regulate translation?

Answer 25

Nutrient and Amino Acid Sensing

Energy State Sensing

Growth Factor Response

Question 26

How does mTOR react to nutrient supply?

Answer 26

Through Amino Acid level sensing.

The amino acid level itself is sensed by a protein known as X, which activates Ras which in turn activates Rheb to stimulate mTOR.

mTOR can also respond to the level of amino acids by regulating the amino acid transporters through an unclear mechanism.

Question 27

How does mTOR react to energy state (AMP:ATP) in the cell?

Answer 27

A high proportion of AMP activates AMP Kinase (AMPK) which phosphorylates TSC2, which is part of a complex with TSC1. This complex forms a GTPase that hydrolyses the GTP bound to Rheb to GDP causing it to inhibit rather than stimulate mTOR.

By this mechanism translation is supressed when little energy is available

Question 28

How do growth factors regulate translation through mTOR?

Answer 28

The growth factors are recognised by receptor tyrosine kinases (RTKs) which activates PI3K (phosphatidylinositol 3-kinase). PI3K phosphoactivates PDK1 which in turn phosphoactivates protein kinase B (PKB).

Protein kinase B phosphoinhibits the TSC complex.

TSC1 inhibits the Rheb, PKB inhibition of TSC1 therefore increases its stimulation of mTOR.

Question 29

How do growth factors increase translation not through mTOR?

Answer 29

PDK1 phosphoactivates S6K as well as PKB, but S6K activates translation through a mechanism that does not involve mTOR.

They can also activate MNK through an entirely different pathway, which has different effects on translation.

Question 30

How can growth factor signalling to translation be inhibited?

Answer 30

By PTEN, which phosphoinhibits PI3K.

Question 31

How can growth factors and stresses can be used to upregulate translation through the action of MNK?

Answer 31

Growth factors and mitogens act through Erk and stresses through the MAPK pathway, but how the MAPK pathway is activated is unknown.

MNK binds the initiation factors eIF4E and G. When phosphorylated MNK phosphorylates eIF4E allowing them to dissociate and so be involved in translation.

Question 32

In what way is translation regulated by insulin?

Answer 32

During times of high insulin levels the expression of GLUT4 glucose transporter protein is increased. As well as upregulating its transcription this is an example of where the effect is on the translation of the mRNA.

Question 33

What is the mechanism of GLUT4 translation regulation?

Answer 33

This is done without need for an extra signalling pathway because the insulin signalling pathway involved stimulation of the PI3K – PDK1 – PKB pathway, so the stimulation of PKB acts not only to stimulate the existing GLUT4 containing vesicles to deliver the receptors to the cell surface but also to increase the rate of their translation and transcription.

Question 34

How is cyclin B1 tranlastion repressed?

Answer 34

It is repressed during the S-phase at the initiation step by a protein called Maskin, which binds to a protein called CPEB which is itself attached to a 3’-UTR region called CPE. Maskin serves to link this to eIF4E – the cap binding protein, circularising the mRNA incorrectly before initiation can begin.

Maskin sequestration of eIF4E is used similarly in many other processes, and is well documented as a key part of early development in Xenopus.

Question 35

How is Cyclin B1 translation re-activated during the M-phase?

Answer 35

the Maskin is dislodged from the eIF4E by PABP, which takes its place and circularises the mRNA properly for initiation to begin.

Question 36

How is translation of genes involved in iron metabolism repressed?

Answer 36

By a protein called Iron Regulatory Protein (IRP) which binds the mRNA shortly after the Cap complex and shapes it into a hairpin loop within the protein body, thus preventing ribosome binding.

Question 37

Why is translation regulated during viral infections?

Answer 37

During viral infection the body inhibits general translation to prevent production of viral proteins.

Question 38

How is translation regulated during viral infections?

Answer 38

By interferons, which are directly stimulated by the infection. Interferons are secreted by the infected cell and bind to other cell’s surface receptors triggering antiviral and anti-translation genes such as RNaseL and PRK (Protein kinase RNA-activated). RNaseL degrades the mRNA and PRK phosphorylates eIF2A, inhibiting initiation of translation.

Question 39

What is different about picornovirus protein translation?

Answer 39

Most viral mRNA has the same 7-methyl guanosine cap structure, but in the picornovirus transcript it is not present. This means the viral mRNA relies on the IRES mechanism for translation.

Question 40

How doe picornovirus upregulate translation of its own genes?

Answer 40

The virus causes cleavage of eIF4G, preventing translation through cap-dependent initiation – which is used for the majority of cellular transcripts. IRES does not use all of eIFG4, requiring only the C-terminal fragment to recruit the 40S subunit through interaction with eIF3.

This means that the viral transcripts are translated much more often as only the IRES initiated mRNAs are initiated.

Question 41

How does initiation begin for Hep C transcripts?

Answer 41

Hepatitis C transcripts require only eIF3 for recruitment of the 40S ribosomal subunit.

When it does bind and identify the AUG by IRES the 40S undergoes a dramatic conformation change that places the AUG at the P site.

Question 42

Where do various antibiotics that target translation act?

Answer 42

• Puromycin is an aminoacyl-tRNA analog, causing premature termination.
• Tetracycline inhibits binding of aminoacyl-tRNA to the A site.
• Chloramphenicol inhibits peptidyl transferase activity.
• Erythromycin binds to peptidyl transferase and prevents polypeptide translocation.

Question 43

How can misregulation of the translation control pathways lead to cancer?

Answer 43

PTEN and the TSC1/2 complex are both tumour supressing proteins whose inactivation leads to cancer as when mTOR is not inhibited it causes proliferation and protein synthesis.

Damage to the TSC1/2 genes leads to hamartomas, also called Tuberous sclerosis, a benign tumour growth of varying severity. They most commonly appear in the brain, kidneys or skin and can present at any age.