Eukaryotic Translation

What is the translation model for translation initiation?

• 43S initiation complex binds 5’ cap

• Scans 5’ → 3’ until first AUG is selected

• 60S subunit joins, elongation starts

What are the subunit sizes in eukaryotic ribosomes?

• 40S, 60S

Simply, what two pieces of evidence verify the whole scanning model?

• Evidence that 90% mRNAs initiate at first AUG

• Evidence that 5’ cap is used for initiation

What evidence is there that the first AUG is selected during the scanning model of initiation?

• 90% of mRNAs initiate at the first AUG

• Inserting new AUG between '5’ cap and original initiation site causes initiation to begin at the new inserted AUG

• Insertion of hairpin loop between 5’ cap and first AUG inhibits translation, so must be translocation between the two

What evidence is there that the 5’ cap is used in initiation?

• Uncapped mRNA are translated accurately but inefficiently

• Addition of cap analogue reduces translation efficiency

• Insertion of hairpin loop between 5’ cap and AUG inhibits translation, so must be translocation between the two

Summarise what the three major steps in translation initiation are

• 43S initiation complex

• Cap-binding complex forms at 5’ of mRNA

• Assembly of 48S initiation complex and scanning

Describe fully the assembly of the ribosome in translation initiation

• 40S subunit associates with eIF3 and GTP-bound eIF2 into 43S complex

• eIF4A,G and E associate together into cap binding complex eIF4F, assembles on cap

• 43S binds cap binding complex

• Scans mRNA until first AUG

What happens when the first AUG is found by the 48S initiation complex?

• Helicase activity of eIF4A unwinds initial secondary structure using ATP hydrolysis

• eIF2-GTP is hydrolyzed, GDP released

• Causes release of all initiation factors except eIF4G

• 60S recruited

• eIF4G binds polyA binding proteins at 3’

• Interaction stimulates transition into elongation

eIF4A

• Helicase upon first AUG encounter

eIF4G

• Binds polyA binding proteins in transition from initiation to elongation

eIF3

• Prevents reassociation of ribosomal subunits

• 10x less abundant than ribosomes so only 10% ribosomes interacted with

eIF2

• Binds GTP

• GTP hydrolysis when eIF4A helicase activity activated

• GDP release causes release of all initiation factors except eIF4G

What evidence is there that eukaryotic mRNAs are monocistronic?

• Ribosomes scan starting at 5’ cap and eIF4A helicase activity unwinds the first AUG, therefore cannot begin at downstream AUGs that would theoretically be other cistrons as scanning has already stopped and ribosome has assembled

How do picornaviruses interfere with translation?

• Uncapped mRNA has long 5’ UTRs with many AUGs and complex, stable, secondary structures, including IRES

• All prevents access to authentic AUG

How do polioviruses interfere with translation?

• Encode proteases that cleaves eIF4G

• Prevents formation of cap-binding complex

• Has an IRES, so can bind to ribosome cap-independently

• Shuts off efficient host translation whilst promoting its own translation

What evidence is there for the action of IRES

• Dicistronic reporter, where complete translation of cistron A causes ribosomes to detach so cannot translate cistron B

• Insert IRES between cistrons A and B

• B is now translated

When regulating translation, which stage should be targeted and why?

• Initiation

• Would be wasteful to get partly through elongation just to terminate

• Would have to degrade what is formed of polypeptide

• Could be risky if partially formed polypeptides happen to be analogues of ligands to receptors or interfere with other cellular pathways before they are degraded

Where does translation occur?

• Cytoplasm and rER surface

Why is regulation of translation via phosphorylation of eIF2-GDP useful?

• Binds eIF2B and sequesters it

• Very little eIF2B present in cell, so effects of sequestering seen rapidly

Describe fully how eIF2-GDP can be modulated to regulate translation?

• When eIF2 is bound to GTP, it can bring the initiator tRNA to the ribosome as part of the cap binding complex

• Its hydrolysis releases it as well as most other initiation factors so the complex can enter elongation

• eIF2-GDP is recycled using guanine exchange factor eIF2B

• Phosphorylation of eIF2-GDP causes binding to eIF2B, sequestering it and preventing it from recycling the GTP of both that factor and others

What part of the host anti-viral response is activated to prevent translation of viral RNA?

• Activation of protein kinase R

• Phosphorylates eIF2-GDP

• This binds eIF2B to prevent guanine exchange

• Prevents translation

How can RNA binding proteins regulate translation?

• Gene specific

• Bind specific sequences in mRNA, then use an intermediate sequence-specific bridging protein to form a loop between polyA site and proteins bound to 5’ cap e.g. eIF4E

• Inhibits recruitment of the rest of the cap-binding complex

Describe mRNA decay

• Ribonucleases, which can be exo and endo, and function both 5’ → and 3’ → 5’

• Transcript specific

• Require both cis and trans factors, so highly regulated

How can cellular mRNA levels be changed?

• Modulating transcription rates, decay rates, or both

How can mRNA degradation be measured experimentally?

• Block transcription of the gene by inhibiting RNAP II or cloning gene of interest under regulatable promoter

• Changes in mRNA levels are then reflective of decay rate as there is no synthesis

Simply, what are the two non-specific mRNA decay pathways? What happens when one pathway is inactivated?

• Decapping pathway

• 3’ → 5’ exosome pathway

• Relatively small change as pathways are mostly redundant

Describe the decapping pathway of mRNA decay - is it specific or non-specific?

• Non-specific

• Lsm protein binds shortened polyA tail of aged/cleaved mRNA to promote decapping

• Can now be degraded 5’ → 3] by exonuclease XRN1

Describe the 3’ → 5’ exosome pathway of mRNA decay - is it specific or non-specific?

• Non-specific

• Exosome functions 3’ → 5’

What cis elements and trans factors are required by transcript-specific decay?

• Cis: 3’ UTR stabilizing or destabilizing secondary structures e.g. AU rich elements

• Trans: miRNAs, sequence specific RNA binding proteins

Describe transcript specific mRNA decay

• AU rich element cis factors destabilise the mRNA

• Recognized by 3’ → 5’ exosomes

• Recognised by RNA-binding protein trans factors that can recruit the exosome

Describe the importance of mRNA localization?

• Can be localized to specific subcellular compartments

• Asymmetric distribution of encoded proteins

• Essential in neuronal function for independent regulation of protein expression between the axon and the dendrite

Describe the experiment that visualises mRNA localization?

• ASH1 asymmetrically localises to budding tip of S. Cerevisiae

• Fix yeast

• Incubate with fluorescently labelled probe complementary to ASH1 mRNA

• Visualize with confocal laser scanning microscopy

• Can see RNA containing ASH1 3’ UTR localised to bud

Describe four ways of regulating translation, and whether they target transcription (indirect) or translation (direct)

• eIF2-GDP phosphorylation - direct

• Gene-specific RNA binding proteins direct

• Decay - indirect

• mi/siRNAs - indirect

What is mRNA localization an example of?

• Regulation of gene expression

What type of complementarity do miRNAs have and what sort of mRNA degradation is caused?

• Imperfect

• Recruits proteins that destabilize mRNA and repress its translation

What type of complementarity do siRNAs have and what sort of mRNA degradation is caused?

• Perfect

• RISC cleavage

• Rest undergoes standard mRNA pathways e.g. exosome

Where are miRNAs derived from in animals?

• Genome

• pri-miRNA transcribed via usual pathway via RNAP II

Where are siRNAs derived from in animals?

• dsRNA from virus e.g. rotavirus

• Synthetic duplexes introduced experimentally

• Endogenously when normal transcription has given rise to mRNA with extensive hairpins

Describe the full process of miRNA generation in animals (no action on mRNA needed)

• pri-mRNA transcribed via usual RNAP II pathway

• Transcript folds into hairpin

• Hairpin recognizable by Drosha:DGCR8 in the microprocessor complex

• Cleaves

• Releases pre-miRNA

• Translocate to cytoplasm

• Dicer:TRBP2 cleaves further into dimer with 5’ monophosphates and 3’ two nt overhangs

• Loaded onto RISC

• Selective removal of passenger strand

What Drosha and Dicer complexes are involved in miRNA generation in animals?

• Drosha:DGCR8 (microprocessor complex)

• Dicer:TRBP2

Describe miRNAs as a duplex

• 5’ monophosphates

• 3’ two nt overhangs

• Rest is bp’d RNA duplex 21-22nt

Describe the full process of siRNA generation in animals (no action on mRNA needed)

• RNA hairpins introduced into cytoplasm (virus, experimental, endogenous)

• Dicer:DGCR8 cleavage into 20-25bp fully bp’d RNA duplexes

• Loaded onto RISC

• Selective removal of passenger strand

Describe siRNAs as a duplex

• 20-25 fully bp’d RNA duplex

Describe the action of miRNAs once they are loaded onto risk and the passenger strand is removed

• Bind 3’ UTR of mRNA with imperfect complementarity

• Imperfect means exonuclease domain of RISC is not in the correct position for cleavage

• Instead, recruits multiple proteins that destabilizes mRNA and represses its translation

Describe the action of siRNAs once they are loaded onto risk and the passenger strand is removed

• Bind anywhere on mRNA with perfect complementarity

• Perfect means exonuclease domain of RISC is in correct position to undergo conf. change that positions it for cleavage

• Cleaves

• Rest of mRNA degraded by standard mRNA decay pathways e.g. exosome

Where do miRNAs bind mRNA?

• 3’ UTR

Where do siRNAs bind mRNA?

• Anywhere

What is the advantage of using experimental techniques such as RNA/DNA footprinting, RIP, ChIP etc

• Can begin experiment in vivo for protein binding, so when continuing in vitro it doesn’t matter if the environment remains physiological

• Genome wide, can see coordination of genes or localization of protein action

• Fast, efficient analysis of whole genome

• Can be elaborated using mutagenesis to see specific binding elements