Lucie Bartoníčková

1. Lucie Bartoníčková ZIB seminar 27th October, 2008

2. P-bodies – mRNA for degradation + translation repression mRNP granules (byproducts of mRNA metabolism) S.cerevisiae (yeast,mammals) C.elegans - germ cells (also Drosophila, amphibians) human cell culture (mammals) rat hippocampal neurons (mammal neurons) chicken fibroblast eukaryotic mRNA – subcellular localizations: polysomes - translating mRNA stress granules - mRNA stopped intranslation initiation (= „a place to die, a place to sleep“) (P granules) (Wickens, Science 2003)

3. interaction with viral life cycles P-bodies accumulation of some viral RNAs/proteins stress granules Are P-bodies and SGs important for viral life cycles? Or for limiting viral infection? How RNA viruses segregate replication & assembly from translation?

4. stress granule P-body CYCLING OF EUKARYOTIC mRNA polysomes (Parker&Sheth, MolCell 2007)

5. P-BODIES (processing bodies) cytoplasmic foci: aggregates of translationally repressed mRNPs translation repression & mRNA degradation gene silencing conserved core proteins: • mRNA decapping machinery general repression / decay machinery • deadenylase complex additional proteins: • mi/siRNA repression factors (RISC) species/condition specific: • RNA binding prot-s + translation repressors • nonsense-mediated decay (NMD) proteins = degradation of improperly processed mRNA (premature stop-codons) proteins affecting viral function - e.g. antiviral APOBEC deaminase

6. DEGRADATION OF EUKARYOTIC mRNA deadenylation-dependent pathways Ccr4p/Pop2p(Caf1)/Not cx major cytoplasmic deadenylase 1) deadenylation P-bodies 2a) 2b) 5´→ 3´exonuclease proteins involved in decapping Ski cx decapping cx (= cx of 3´→ 5´ exonucleases) 3´→5´ degradation decapping + 5´→ 3´ decay predominant in yeast predominant in mammals (adapted from Parker&Sheth, MolCell 2007)

7. GENE SILENCING miRNAs siRNAs (= microRNAs) (= short interfering RNAs) = dsRNase ~ 21- 23 nt RNA-induced silencing complex RNA interference 3ˇuntranslated region of target mRNA destruction of target RNA translation repression (Lodish et al.,5th ed., adapted from Hutvágner& Zamore 2002)

8. * = P-body components GENE SILENCING & P-bodies miRNAs siRNAs may target mRNAs into P-bodies mRNA decay * * * * * * translation repression mRNA decay(mainly in plants) * AGO = Argonaute proteins – essential components of RISC charactericticdomains: PAZ & PIWI (similar to RNase-H domain) (Eulalio, Nat Rev Mol Cell Biol 2007)

9. STRESS GRANULES transient cytoplasmic bodies induced upon environmental stress (response to defects in translation initiation) contain aggregates of mRNA + translation initiation factors 48S preinitiation cx: eIF4 subunits, 40S ribosomal subunits, poly(A)binding protein 1 (PABP-1) RNA binding proteins with self-interaction domains (TIA proteins) often associated with P-bodies ? mRNA moving between the compartments

10. P-bodies& VIRUSES effects of mutations in various core P body components on viral life cycles

11. a) retrotransposons and P-bodies retrotransposons form virus like particles model: yeast Ty1 (copia-like family) & Ty3 (gypsy-like) may require P-bodies for life cycle: Δs in several prot-s promoting P-body formation reduced retrotransposition,altered subcellular distribution of Ty3 proteins → pop2Δ(deadenylase cx) → enhanced retrotransposition ? role in assembly/maturation of Ty VLPs tagged Ty3 RNA & proteins accumulate in P-bodies precise function still unclear (Roth, Yeast 2000) Ty element life cycle

12. b) retroviruses and P-bodies HIV cellular proteins:Crm1p & RNA helicaseDDX3 - required fornuclear export of unspliced HIV-1 RNA →possible recruitment of HIV-1 genomic RNA to P-bodies for packaging? (Crm1p required for export of P-body components) other retroviruses localisation of viral components (Gag, Pol) to discrete cytoplasmic foci = ?? P-bodies

13. c) + RNA viruses brome mosaic virus (studied in yeast – complete viral life cycle) tripartite genome: RNA1, RNA2, RNA3 – capped, lack poly(A) 1) P-body components(generally translation repressors) required for RNA1-3 translation WHY? 2) P-body components required for RNA1-3 replication (membrane-bound complex) - concentrating genomic RNAs+proteins- promoting interaction with membranes HCV HCV core protein colocalizes in cytoplasm foci (? P-bodies) HCV replication enhanced by interaction with liver-specific miRNA ? P-body components important for efficient HCV replication ?

14. antiviral APOBEC proteins - accumulate in P-bodies & SGs (during stress) x (HIV-1 Vif protein → APOBEC3G degradation) P-bodies & stress granulesinANTIVIRAL DEFENCE miRNAs may recruit P-body components to target mRNAs siRNAs → translation repression + mRNA degradation = cytidine deaminases (apolipoprotein B mRNA-editing enzyme) (x retroviruses, retrotransposons) transientSGs formation triggered by some viral infections SGs may limit viral infections(e.g. VSV ((-)RNA), Sindbis v.(+RNA), HSV (DNA), polio) xsome viruses interfere with SGs formation

15. Host defence or host defeat? P-bodies & stress granules– positive x negative influence on viral life cycles host defence: repressing function of viral transcripts promoting viral life cycle: • viral transcription • nuclear-cytoplasmic transport + remodeling of viral RNPs • concentration of mRNAs - ? recruitment of viral mRNAs for translation, replication, assembly

16. Thank you for your attention!