Seminar 1 Components and Regulation of Initiation of Translation Michael Altmann FS 2015

Institut für Biochemie und Molekulare Medizin

Seminar 1 -  What are the biol. consequences of mRNA transport and localized translation?

-  Inform yourself about CCA adding enzyme (tRNA)!

-  Why are there only around 40 different tRNAs, if 61 codons encode for an amino acid?

-  How many rRNA genes does a cell need to make 106 ribosomes in 6 hours?

-  What are the most prominent differences in the mechanism of initiation between prokaryotes and eukaryotes?

-  What‘s about translation in mitochondria?

-  Why should a cell need so many helicases?

-  How are protein-protein interactions measured?

-  What do you know about G-proteins (function, subunits, mechanism)?

-  Why are 10-20% of eIF2α-P sufficient to block translation?

-  Physiological consequences of translational regulation of transcription factors?

-  Viruses fight against eIF2 kinases! How?

-  What do you know about the roles of small RNAs in gene expression?

-  What would be an advantage in cancer therapy of inhibiting eIF4E‘s-activity with Mnk-repressors?

What are the biol. consequences of mRNA transport and localized translation?

• Synthesis of protein in place where it is needed

• Example: nerve cells. Synapse.

• Programming of cells

• Example: oocytes and maternal mRNA. Yeast: buds

Inform yourself about the CCA adding enzyme (tRNA)!

• CCA is essential for aminoacylation and ribosome-binding of tRNA. Archaea and

eukaryotes have structurally different enzymes.

• Nucleotidyltransferase (CTP, ATP), template-independent. One single catalytic center.

• Mechanism: unknown. 3D structures solved.

Wobbling of the anticodon

• tRNAs contain many post-transcriptional modifications (e.g. methylation of bases, endonucleolytic cleavage) • inosin which is found in tRNAs (e.g. at the first position of the anticodon) is formed by desamination of adenin • wobble = inosin can form base pairs with A, C and U-> only 1 tRNA exists which forms base pairs with 3 related codons which differ in the third position (GCU, GCC and GCA). Wobbles do not exist in the first and second position of codons.

- Why are there only around 40 different tRNAs, if 61 codons encode for an amino acid?

- How many rRNA genes does a cell need to make 106 ribosomes in 6 hours?

Transcription of rRNA genes"

• About 50 RΝΑ-polymerase I molecules synthetize in tandem one transcription

unit (1 new polymerase molecule jumps on rDNA gene each 2 seconds).

- How many rRNA genes does a cell need to make 106 ribosomes in 6 hours?

• Number of nucleotides per ribosome? About 5400 nt.

• Speed of transcription? About 50 nt per second or 108 sec / rRNA molecule.

• G1 phase? 6 hr (21600 sec) -> 200 molecules rRNA / RNA-polymerase I

molecule. About 10'000 rRNAs per rDNA gene.

• For 1 million rRNAs: 100 rDNA genes required.

• About 50 RΝΑ-polymerase I molecules synthetize in tandem one transcription

unit (1 new polymerase molecule jumps on rDNA gene each 2 seconds).

Molecule Velocity Accuracy Correction functions

DNA 600 nt /sec (bacteria) 100-200 nt / sec (eukaryotes)

10-9 - 10-10 • DNA-pol I/II/III 3’-5’ exo; 5-3’ exo (10-7) • postreplicative correction systems (10-3) e.g. uracil-DNA-glycosylase or uvr-system (nucleotide-excision-repair)

RNA 500 nt - 50 nt / sec

10-4 No exo-activity known

Protein 20 aa / sec 10-3 – 10-4 • aa-tRNA synthetases (editing site; 10-4 -10-5!) • eEF1A-GTP-aa-tRNA (kinetic proofreading; 10-3-10-4)

Macromolecular synthesis: velocity and accuracy

What are the most prominent differences in the mechanism of initiation between prokaryotes and eukaryotes?

• coupling of transcription and translation • only 3 initiation factors • tRNA-Meti is formylated to bind at P-site • Shine/Dalgarno sequence • internal initiation

Are there orthologs of eIF’s?

Eukarotes Prokaryotes Archaea

eIF1 IF3 (CTD) aIF1

eIF1A IF1 aIF1A

eIF2: α, β, γ aIF2: α, β, γ

eIF4A IF4A/W2 aIF4A

eIF5B IF2

Translation initiation in bacteria

Reference: Simonetti et al (2009) Cell. Mol. Life Sci. 66, 423-436

Why should a cell need so many helicases?

• Base-pairing and melting • Reactions depending on RNA-RNA interactions • RNA chaperone • Protein-RNA interactions and their resolution

How are protein-protein interactions measured?

• 2-hybrid system (yeast)

• Tagged proteins on solid matrix

• Co-precipitation (antibodies)

• Complexes on sizing columns

• Surface Plasmon Resonance

In complex mixtures:

• FRET

• Co-localization

Why are 10-20% of eIF2α-P sufficient to block translation?

• eIF2B is only 10-20% as abundant as eIF2

• stable complex leads to sequesteration

Physiological consequences of translational regulation of transcription factors?

• Translation controls transcription

• Programming of cells

• Multi-level control

Viruses fight against eIF2 kinases! How?

Inhibition of the kinases:

• Production of high concentrations of dsRNA which inhibit kinase (Adenovirus)

• Production of dsRNA-binding proteins (HSV, Vaccinia, Reo, Influenza)

• Production of pseudo-substrate kinase-inhibitor (K3L of Vaccinia Virus)

• Activation of cellular kinase-inhibitor (p58 by Influenza Virus)

Activation of eIF2:

• Activation of PP1alpha (dephosphorylation of eIF2)

What do you know about the roles of small RNAs in gene expression?

• snRNP and splicing

• RNA modification: snoRNA

• mRNA translation and half-life (gene silencing)

-  What would be an advantage in cancer therapy of inhibiting eIF4E‘s-activity with Mnk-repressors?

Mnk 1 phosphorylates eIF4E when sitting on eIF4G (see graph). Phosphorylation of eIF4E increases its oncogenic properties (shown in several studies) but phosphorylation of eIF4E is not essential for cell survival (shown with ∆mnk1/2 knockouts). -> Mnk-repressors should help to reduce eIF4E activity without compromising cell survival.

Paper presentation An RNA biosensor for imaging the first round of translation from single cells to living animals James M. Halstead et al. (2015) Science 347, 1367-1371

Scheme of TRICK assay

TRICK = Translating RNA Imaging by Coat Protein Knock-Off

Reference: Popp & Maquat (2015), Science 347, 1316-1317

• Two engineered fluorescent proteins which bind to specific RNA-motifs:

MS2 coat protein (MCP) fused to red fluorescent protein (RFP)

PP7 coat protein (PCP) fused to green fluorescent protein (GFP)

• Both proteins carry a Nuclear Localization Signal (NLS) ensuring the return after the first

round of translation to the nucleus.

• Using imaging and computer software, the localization of translated mRNAs can be observed.

Reference: Halstead et al. (2015) Science 347, 1367-1370

RNA biosensor for imaging translation

Validation of system

E) = untreated cells

F) = CHX treated cells

G) = puromycin treated cells

E) = Cytoplasma of a cell translating at high ratio (>90% mRNAS engaged)

F) = Cytoplasma of a cell treated with cycloheximide (<10% mRNAS engaged)

G) = Cytoplasma of a cell treated with puromycin (<10% mRNAS engaged)

Reference: Halstead et al. (2015) Science 347, 1367-1370

RNA biosensor for imaging translation 1: Does translation occur in the nucleus?

U-2 OS cells expressing TRICK reporter

• Only yellow dots are observed in the nucleus which is

surrounded by cytoplasma with red dots

Conclusion 1

• No nuclear translation can be observed

• Ribosome profiling shows that mTOR regulates the translation of 5‘TOP, 5‘TOP-like, PRTE (Pyrimidin Rich Translational Element) containing mRNAs. Hsieh et al. (2012) Nature 485, 55-61 Thoreen et al. (2012) Nature 485, 109-113

• Are there proteins binding to 5‘TOP sequences involved in regulating mRNA translation?

Gentilella & Thomas, N & V (2012) Nature 485, 51-52

RNA biosensor for imaging translation 2: Are 5‘ TOP mRNAs accumulated in stress particles?

What are P-bodies & stress granules?

• Stress (such as arsenite treatment) leads to inhibition of ternary complex (eIF2•GTP•Met-

tRNAiMet) and the formation of

P(rocessing)-bodies

Compartments in eukaryotic cell where mRNAs are located and translationally silenced during

stress.

P-bodies carry 5‘- and 3‘nucleases which can result in mRNA degradation.

Here: immunofluorescent P-bodies (red)

• Both are dynamic structures (form and desintegrate depending on conditions)

none of both compartments carries membranes

Stress granules

Compartments in the cytoplasma where preinitiation mRNA complexes are sequestered during

stress.

Here: immunofluorecent stress granules (green)

Reference: Halstead et al. (2015) Science 347, 1367-1370

Stress applied to HeLa cells: 60 min 0.5 mM arsenite treatment

RNA biosensor for imaging translation 2: Are 5‘ TOP mRNAs accumulated in stress particles?

Conclusion 2A

• Due to stress, 5‘TOP but not ∆5‘TOP mRNAs accumulate in P-bodies

Reference: Halstead et al. (2015) Science 347, 1367-1370

Removal of arsenite stress

RNA biosensor for imaging translation 2: Are 5‘ TOP mRNAs accumulated in stress particles?

Conclusion 2B

• Upon removal of arsenite stress, 5‘TOP mRNAs in P-bodies remain untranslated

• In Drosophila, localized expression of Oskar protein at the posterior pole of the oocyte is

essential for correct body patterning and germ cell formation.

RNA biosensor for imaging translation 3: Can pole-specific translation of oskar protein be visualized?

• Before reaching the posterior pole, oskar mRNA translation is inhibited by Bruno, a protein

which binds to the 3‘UTR of oskar mRNA and recruits Cup, an eIF4E binding protein. This

complex is translationally silenced.

• Later in oogenesis, osk mRNA translational repression is alleviated in the posterior pole.

Reference: Halstead et al. (2015) Science 347, 1367-1370

RNA biosensor for imaging translation 3: Can pole-specific translation of oskar protein be visualized?

Translation of oskar mRNA:

In early stage oocytes oskar mRNA binds both MCP and PCP -> translationally inhibited.

Conclusion 3

During later stages, the PCP signal is reduced and Oskar protein is detected in the posterior

pole-> oskar mRNA is translationally active.

TRICK reporter: osk mRNA carrying 12xPP7 in the ORF and 6xMS2 stem-loops in the 3‘UTR

Take home message

The TRICK reporter system offers a spatial and temporal visualization of active

translation as shown for

• Nuclear translation

• TOP mRNAs in P-bodies

• oskar mRNA translation in the posterior compartment of Drosophila oocytes

Diffusion of mRNA molecules in the nucleus