L1:

The central Dogma

• Coding region of mRNA specifies what protein is going to be produced

• Ribosomes translate mRNA into proteins, translation factors promote this pathways & tRNA deliver the relevant AA in the right order

• Amino acids are the raw materials used in translation

• Uses ATP & GTP hydrolysis to provide E for process

• Multiple surveillance pathways to make sure protein production & reading the code is done accurately - makes sure process only occurs when needed and when E is available

Ribosome composition

• An RNA scaffold coated by proteins

Ribosome - History & background

• 1970s → biochemical investigation of translation

• 2000 → 1st high resolution structure of ribosome (x-ray) by Venki Ramakrishnan

• 2000 onwards → dozens of structures of various functional state of the ribosome - rationalisation of more than 30 yrs of biochemical findings

• 2009 → Nobel Prize (chem.) to Yonath, Steitz & Ramakrishnan for their structural investigations of the ribosome

  → translation pathways deciphered (initiation-elongation termination)

  → antibiotics mode of action unravelled

Ribosomes

• Many RP paralogs produced in higher eukaryotes

• RPs can be post-translationally modified

• Variable rRNA modification

• Variability in composition → specialised ribosomes

Ribosomes - protein synthesis in 3 steps

mRNA features that influence eukaryotic translation

• Eukaryotic DNA capped at 5’ end w/ m7G cap → protective for degradation & crucial for translation

• ORF contributes to protein code being produced - section from start codon to end codon

• 3’ poly(A) tail contributes to translational control

• Most eukaryotic translation is cap dependant

Translation initiation: cap binding

• Cap binding complex (eIF4F complex) which helps signify the 5’ end

• eIF4F complex → 3 diff. proteins that contribute to cap recognition

  • eIF4E → high affinity for the cap & binds it directly

  • eIF4G → bigger protein which binds 4E

  • eIF4A → associated w/ 4G & its an RNA helicase

• Poly(A) tail on 3’ end - bound by poly(A)-binding protein which interacts w/ eIF4G & helps stabilise 4F complex & binding at the 5’ end

Translation initiation: ribosome recruitment

• Small ribosome (40s) forms a pre-initiation complex w/ other initiation factors

  • eIF3 directly binds 4F & the ribosome → help bridge & bring that ribosome in

  • eIF1 & 1A have proofreading roles

  • eIF2 part of a Ternary complex which comes in & delivers initiator tRNA (tRNA loaded w/ initiator methionine) & comes in w/ eIF5 → provides GTPase activity (release E)

• 43S initiation complex brought in to 5’ prime end of mRNA through bridging interactions of eIF3

Translation initiation: scanning

• To find the correct start codon, it scans along the mRNA, 5’ to 3’ until it finds AUG

• eIF4A is helicase used to unwinds RNA structure to be scanned properly

• Use E through ATP hydrolysis

• Recognition of AUG is evident through base pairing

  • anticodon of initiator tRNA base pairing w/ AUG codon

• Once scanning ribosome has located AUG → 48S complex

Translation initiation: AUG recognition

• This triggers release of eIF5 & eIF2 - using ATP hydrolysis

• eIF1 & 1A - important for locating correct AUG → to avoid frame shifts

  • by sitting at correct interface b/w tRNA anticodon & mRNA

Translation initiation: 80S assembly

• Joining of 60s large subunit w/ 40s→ facilitated by eIF5B

  • using GTP hydrolysis

• Ejection of remaining initiation factors - not needed anymore

Translation initiation complete

• 80s ribosome also known as monosome → ready to elongate

Comparison of eukaryotic & prokaryotic translation

L2:

Translation control & disease

• Rate limiting steps are key targets for regulation & dysregulation

eIF4G structure & regulation during apoptosis

• In normal cells, eIF4F complex helps to recruit 43s ribosome pre-initiation complex - through interactions that eIF4G has w/eIF3

• eIF4F complex also interacts w/ poly(A)-binding protein which is bound at the 3’ end

  • communication b/w 5’ & 3’ helps stabilise the 4F complex association w/ mRNA & promote translation

• When all interactions proceed, cap-dependant translation occurs

• Apoptosis→ important in development - to get rid of cells that are no longer needed & in normal tissue homeostasis to eliminate infected cells

• During apoptosis - there is activation of caspases (enzymes that can cut other proteins)

  • they proteolytic cleave eIF4G in 2 positions → cuts separate the parts of the proteins that interact w/ protein interactors that promote translation initiation

  • left w/ middle fragment (p76) that interacts w/ eIF4E, but not w/ poly(A)-binding protein & impaired in ability to promote translation initiation

  • ∴ disruption of cap-binding complex activity & overall deregulation of translation in apoptosising cells

• Some specific messages that are required to proceed w/ apoptosis, need to be produced

  • through alternate mechanism → cap-independent translation by internal ribosome entry site (IRES) - e.g. XIAP (inhibitor of apoptosis)

IRES dependant translation

• IRES → structure RNA domain that recruits the ribosome

  • facilitates recruitment of a ribosome internally to the mRNA - rather than recruiting a ribosome directly to the 5’ cap

• No need for the cap recognition & scanning - don’t need eIF4E binding for the cap

• Require only few eIFs, specific subset varies w/ each IRES

• Contains stem loops → highly stable & helps recruit in these proteins

• e.g.XIAP → IRES recruits in eIF3 directly

  • to allow positioning of 40s ribosome directly onto the AUG

  • mediates 5’ -3’ communication the same way 4G does

• Present in both cellular mRNAs & viral RNAs

  • evolved in viruses to produce their proteins even when the cell is apoptosing

IRES dependant translation

• An assay used to define the function of an IRES - in place of cistrons - fluorescent proteins of diff . colours

• 2 cistrons (open reading frames that code for reporter proteins) are added to an mRNA

• Positioning 2 open reading frames next to each other in mRNA that’s capped

• In eukaryotic cell via cap-dependant translation

  • 1st cistron will be translated to produce the product & at end of translation, ribosome will terminate & ejected from mRNA protein being made

  • no translation of downstream 2nd cistron

• To test whether a seq. identified in RNA is functioning as an IRES - the sequence is put b/w 2 cistrons

  • 1st cistron - translated in cap-dependant manner → after cap binding complex has recruited the ribosome at cap & allowed 1st cistron to be translated

  • direct recruitment of a ribosome to IRES seq. - if functioning will allow production of 2nd protein → finds out whether seq. can function as IRES & what the requirements are e.g. what eIFs are important for functioning of IRES

• Cap-independent translation → no translation of either cistrons

• Highly stable stem loop added to 5’ UTR → prevents unwinding activity of eIF4A → ∴ no translation from 1st cistron but effective translation from 2nd cistron using IRES

Regulation of cap recognition

• Unstructured 5’UTR → less dependant on 4A activity ∴ not using as much E

• Structured 5’UTR → e.g. stem loops present - require lots of E from 4F complex to unwind & allow translation to occur → poorly translated in cap-dependant translation

Regulation of cap recognition: eIF4E levels

• UTR containing mRNAs disproportionately benefit when 4E levels are higher

  • higher 4E levels → help translate poorly translated mRNAs

  • pro-proliferative factors e.g Cyclin D1 & C-myc contain these - upregulation of their expression may cause uncontrolled growth of the cells

• Cultured cells that have overexpressed eIF4E observe cell transformation (transformation to phenotype resembling cancer)

Regulation of cap recognition: eIF4E sequestration

• Δ availability of eIF4E in cell can be regulated in its activity

  • 4E bound by eIF4E binding protein (4E-BP) - 4e no longer able to bind cap & participate in translation initiation

  • 4e-BP phosphorylation releases 4E for efficient cap binding

• This is regulated through signalling cascade - mTOR signalling pathway →

  • processes mitogenic signals from cytokines, hormones etc. that are interpreted by receptor Tyr kinases

  • through cascade of phosphorylation, culminating in activation of mTOR → results in phosphorylation of 4E-BP activating translation

• 4E can be directly phosphorylated by kinase Mnk → ↑ affinity for cap so functions better

• Mnk activated through MAP kinase pathway - also activated by mitogenic signals

• Signalling pathways can impact the phosphorylation activity of other eIFs e.g.

  • mTOR pathway can also lead to phosphorylation of eIF4A - helicase in eIF4F translation initiation complex & phosphorylation of cofactor 4B

  • phosphorylation of both factors is associated w/ ↑ activity

Targeting translation as cancer therapy

Translational control & disease

Regulation of ternary complex formation

• At the end of translation, eIF2 associates w/ GDP due to GTP hydrolysis during initiation

• eIF2 has 3 subunits: α, β, γ

  • γ is bound to GTP or GDP

• GTP required for translation initiation - associates w/ initiator tRNA & the ternary complex can participate in translation

• eIF2 recycling → eIF2B (GTP exchange factor) required to help promote transfer of GDP to become GTP

  • by directly binding eIF2

• Process of recycling can be regulated by phosphorylation of eIF2 α subunit using eIF2 α kinases

  • when phosphorylated GDP bound eIF2 can’t be recycled into GTP

• Phosphorylated eIF2 binds eIF2B v/ tightly → issue due to less eIF2B in the cell than eIF2

• 4 diff. types of eIF2 alpha kinases in mammalian cells → each processes diff. stress responses:

  • HRI → sense low haem levels & inhibits translation of proteins to reduce production of globins

  • GCN2 → sense low amino acid levels

  • PKR → activated by dsRNA → hallmark of virus infection

  • PERK → senses ER stress - response to presence of unfolded proteins in ER

• Q: why shut off translation when cells are stressed?

• turn off translation when AA are low

  • prevents the wrong AA from being translated

  • prevents E from being wasted on wrong resources

• turn off translation if cells infected w/ virus

  • viruses that infect cells contain own RNA/DNA or enzymes → so stops virus infection by preventing viral cells from using host ribosomes & stopping protein synthesis of viral proteins

PKR structure, function & regulation

• dsRNA is hallmarker for viral infection

• PKR has 1 domain that helps bind dsRNA & kinase domain at C terminus that’s activated when PKR binds the dsRNA → leads to eIF2 alpha phosphorylation → blocks viral protein production

• Viruses have evolved ways to antagonise PKR

  • e.g. viruses e.g. HSV1 Us11 - produce dsRNA binding proteins - shield recognition of dsRNA

  • some viruses may decoy RNAs which are bound by PKR but don’t activate it

  • HCV NS5A - make antagonists of kinase activity of PKR - so they directly stop activity or kinase domain

PKR-like ER localised kinase (PERK)

• Sense unfolded protein buildup on ER → burden on machinery that folds proteins into active form & causes imbalance

• Unfolded proteins may be toxic - need to stop build up

• PERK inhibits protein synthesis so that backlog is reduced

eIF2B mutations cause disease

• Example of failure to fully recycle eIF2 & maintain normal levels of ternary complex is in the disease Vanishing white matter (leukodystrophy)re

  • specifically affects myelinated cells (white matter)

L3:

When translation goes right…

• Recruited ribosome to 5’ end (monosome) & then further rounds of recruitment of ribosomes to 1 mRNA - to maximise production of proteins from 1 mRNA (polysomes)

• Functional protein produced

When translational goes wrong…

• **Premature termination codon (PTC)**→ when a termination codon is present in middle of open reading frame - introduced by transcription error in nucleus or mutation in DNA

  • only makes 1/2 a protein ∴ non-functional & waste of E

  • toxic truncated proteins → e.g. in transcription factors - 2nd part of protein (not coded) may have contained a regulatory domain which helped keep control of enzyme

Nonsense Mediated Decay (NMD)

Features in mRNA that help to identify premature termination codon (PTC):

• **Exon-junction complex (EJC)**→ at exon-exon boundary

  • during pioneer round, ribosome is recruited & as it elongates through open reading frame, it displaces the EJCs along the length of mRNA

  • after pioneer round, no more EJCs & mRNA can be translated efficiently

• When PTC is present - translating ribosome starts elongating along 1st portion of open reading frame & knocks off EJCs - then stops at termination codon to terminate

  • downstream, still EJC present → alarm to ribosome that termination codon isn’t in correct place

    • SURF complex formed due to paused ribosome in proximity of EJC → adaptor to bring in RNA decay effectors that dispose of mRNA that is detected as abnormal

• Triggers a safety net to degrade the truncated polypeptide that’s been generated

• Decay that’s initiated is an endoribonucleolytic cleavage - cuts in middle of RNA

  • complex also recruits SMG6

Other quality control pathways

• Nonsense-mediated decay (NMD) → (premature termination codons) quality control to eliminate problematic mRNA & limit production of toxic proteins in cytoplasm

• No-go decay (NGD) → ribosome stalled in translation mRNAs e.g. it hits a stable RNA loop, rare codon present in mRNA or damage to RNA

  • to rescue stuck ribosome - Pelota & HBS1 are recruited

• Non-stop decay (NSD) → mRNAs w/o natural stop codons e.g. mistake in transcription, mutation in DNA or transcription termination hasn’t happened fully at end of gene ∴ added poly(A) tail to middle of seq. → ribosome starts translating into poly(A) tail

Specific mRNA translation regulation

• Most regulation occurs through 5’ & 3’ UTRs

• When global mechanisms have specific consequences (uORFs)

• How miRNAs & RBPs can regulate translation from 3’ end

• how epitranscriptomic changes can influence translation

Translational repression by a uORF (upstream Open Reading Frames)

• Translation from central ORF is dependant on scanning from 5’ cap of a ribosome & initiating at AUG

• Some mRNAs have short ORF in 5’ UTR - encode for small peptides

  • scanning ribosome initiates at upstream ORF to produce short peptide - at end of translation, ribosome terminates & leaves mRNA

  • downstream ORF isn’t translated ∴ poorly translated as ribosomes won’t reach downstream AUG

  • beginning translation at upstream ORF, inhibits initiation at downstream ORF

    • peptide encoded by uORF can specifically interfere w/ translation at ribosome

Translational control by a uORF (upstream ORF)

• mRNAs w/ upstream ORFs are generally encoding products that post-production needs to be v. tightly controlled & strongly inhibited most of the time

• These examples contain ORF & help ↑ translation when eIF2α is phosphorylated:

  • e.g. GCN4 → transcription factor - activates large no. of genes involved in AA synthesis → most of time - no GCN4 produced as translation inhibited by upstream ORF

    • low AA & eIF2α kinase has lead to phosphorylation of eIF2 complex → ↓ in ternary complex availability that delivers initiator tRNA to ribosome → ribosome initiated on GCN4 mRNA - scans along UTR but misses upstream ORF due to low availability of initiator tRNA to start translation

      • translation improves on uORF containing mRNA from low base level

      • ↑ production of GCN4 helps synthesis AA to get out of cell stress situation

  • e.g. ATF4 → helps produce chaperone proteins to help resolve ER stress → improves translation

  • GADD34 → helps guide phosphatase to dephosphorylate eIF2α

Translational control from 3’ end - RNA-BPs

• RNA binding protein binding to cis-acting element in 3’ UTR can impact on initiation at 5’ cap

• Cis-acting element→ element in RNA which acts on same RNA (on itself)

Translational control from 3’ end- e.g. caudal & bicoid

• Patterning needs to occur so that v. early on in development, embryo knows which end in head & which is tail

  • done molecularly by gradients of proteins (morphogens) that influence this patterning - communicate their fate to cells during embryogenesis

    • e.g. bicoid → gene is selectively expressed at head end (anterior)

      • transcription factor that’s important for production of other patterning mols. & acts as translational regulator on mRNA called caudal

    • e.g. Caudal → important for patterning the posterior end of embryo, but it’s mRNA is expressed throughout the embryo - bicoid stops protein being where bicoid is present

• Patterning the early drosophila embryo:

• Caudal is an important patterning mol. during embryogenesis

• Bicoid (RBP) is RNA binding protein that interacts w/ 3’ UTR of caudal mRNA

• Bicoid recruits 4E-HP (4E homologous protein) to bing caudal cap but not initiate translation

• 4E-HP blocks eIF4F binding & translation of caudal mRNA

Translational control from 3’ end - e.g. msl2 & SXL

• In drosophila, to achieve dosage compensation of X-linked genes, in male flies, the dosage compensation complex (feat. msl-2) is required for hypertranscription (massively upregulate) of single X chromosome

  • mediated by protein msl-2

• msl2 mRNA is transcribed in both, but translated in male but not female during development - to repress production in female flies

• SXL (“sex-lethal”) is only expressed in females & is a binding protein

• SXL & UNR bind to 3’ UTR of msl-2 & interact w/ poly(A) binding protein & influences eIF4F complex to stabilise it’s interaction w/ pol(A)

  • becomes too stable - prevents 4F to recruit 43s ribosome - translation is inhibited on msl2 in female flies

• In early embryogenesis, mutation of SXL will compromise establishment of sex

Translational control from 3’ end - microRNAs

• Regulatory RNA seq. in 3’ UTR that mediates binding of RNA complex & leads to inhibition of translation initiation → occurs prior to mRNA decay & also occurs on microRNA targets

• other example of mech. of control from 3’ UTR

Translational control - beyond the genetic code

• RNA bases can be chemically modified in a way that doesn’t Δ the code → the epitranscriptome

  • influences mRNA fate

  • e.g. methyl-cytosine or methyl-adenosine

• mRNA modifications correlate w/ mRNA features

  • most freq. modification - m7 cap → methyl 7 on guanosine cap

Translational control - N6-methyladenosine (m⁶A)

• m⁶A promotes mRNA translation by multiple mechanisms

• “reader proteins” can bind m⁶A in 3’ UTR & enhance translation initiation e.g YTHDF1 & 3

• METTL3 → helps install m⁶A & adds on m⁶A → promotes translation of message m⁶A is on (cap-dependant translation)

• When m⁶A is present on 5’ UTR it can function like IRES → translation factor eIF3 can be recruited by m⁶A directly

m⁶A controls multiple RNA-regulatory processes

• m⁶A implicated in control of RNA stability & degradation

• Can impact nuclear export & splicing

• Functions are dictated by diff. kinds of BPs that binds the m⁶A modification

• can be demethylated later on → turned on or off → epitranscriptomics

Questions & themes to consider

• What mechanisms exist to ensure accurate translation of error-free mRNAs?

• Which control mechanisms target general translation factors but selectively affect the translation of a subset of mRNAs?

• Which mutations or changes in eIFs are associated w/ disease?

• Which control pathway normally prevent disease?

• How might a circular RNA, lacking a 5’ cap, be translated?