Download RNA biosensor for imaging translation

Institut für Biochemie und Molekulare Medizin
Seminar 1
Components and Regulation of Initiation of
Translation
Michael Altmann
FS 2015
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 Mnkrepressors?
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.
- Why are there only around 40 different tRNAs, if 61 codons encode for an amino acid?
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. - 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.
• About 50 RΝΑ-polymerase I molecules synthetize in tandem one transcription
unit (1 new polymerase molecule jumps on rDNA gene each 2 seconds).
• 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.
Macromolecular synthesis: velocity and accuracy
Molecule Velocity
Accuracy
Correction functions
DNA
10-9 - 10-10
• DNA-pol I/II/III
3’-5’ exo; 5-3’ exo (10-7)
600 nt /sec
(bacteria)
• postreplicative correction
systems (10-3)
e.g. uracil-DNA-glycosylase or
uvr-system (nucleotideexcision-repair)
100-200 nt / sec
(eukaryotes)
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)
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
eIF1
IF3 (CTD)
aIF1
eIF1A
IF1
aIF1A
eIF2: α, β, γ
Archaea
aIF2: α, β, γ
eIF4A
IF4A/W2
eIF5B
IF2
aIF4A
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 Mnkrepressors?
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
• 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:
Popp & Maquat (2015), Science 347, 1316-1317
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
Reference:
Halstead et al. (2015) Science 347, 1367-1370
RNA biosensor for imaging translation
2: Are 5‘ TOP mRNAs accumulated in stress particles?
• 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
What are P-bodies & stress granules?
• Stress (such as arsenite treatment) leads to inhibition of ternary complex (eIF2•GTP•MettRNAiMet) 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)
Stress granules
Compartments in the cytoplasma where preinitiation mRNA complexes are sequestered during
stress.
Here: immunofluorecent stress granules (green)
• Both are dynamic structures (form and desintegrate depending on conditions)
none of both compartments carries membranes
RNA biosensor for imaging translation
2: Are 5‘ TOP mRNAs accumulated in stress particles?
Stress applied to HeLa cells: 60 min 0.5 mM arsenite treatment
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
RNA biosensor for imaging translation
2: Are 5‘ TOP mRNAs accumulated in stress particles?
Removal of arsenite stress
Conclusion 2B
• Upon removal of arsenite stress, 5‘TOP mRNAs in P-bodies remain untranslated
Reference:
Halstead et al. (2015) Science 347, 1367-1370
RNA biosensor for imaging translation
3: Can pole-specific translation of oskar protein be visualized?
• In Drosophila, localized expression of Oskar protein at the posterior pole of the oocyte is
essential for correct body patterning and germ cell formation.
• 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.
RNA biosensor for imaging translation
3: Can pole-specific translation of oskar protein be visualized?
TRICK reporter: osk mRNA carrying 12xPP7 in the ORF and 6xMS2 stem-loops in the 3‘UTR
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.
Reference:
Halstead et al. (2015) Science 347, 1367-1370
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