Lucie Bartoníčková ZIB seminar 27 th October, 2008

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

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

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

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

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

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

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

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

effects of mutations in various core P body components on viral life cycles group virus virus-like element studied in phenotype of mutations retro- trans- posons Ty1 & Ty3yeast reduced retrotransposition Ty3yeast enhanced retrotransposition +RNA viruses brome mosaic virusyeast reduced translation & rectruitment to replication HCV mammalian cell culture reduced replication retro- viruses HIV mammalian cell culture reduced nuclear export and translation of unspliced HIV-1 transcripts P-bodies & VIRUSES

a) retrotransposons and P-bodies retrotransposons Ty element life cycle form virus like particles model: yeast Ty1 (copia-like family) & Ty3 (gypsy-like) may require P-bodies for life cycle: pop2 Δ (deadenylase cx) → enhanced retrotransposition reduced retrotransposition, altered subcellular distribution of Ty3 proteins Δs in several prot-s promoting P-body formation ? role in assembly/maturation of Ty VLPs (Roth, Yeast 2000) tagged Ty3 RNA & proteins accumulate in P-bodies precise function still unclear →

b) retroviruses and P-bodies HIV - required for nuclear export of unspliced HIV-1 RNA → possible recruitment of HIV-1 genomic RNA to P-bodies for packaging? other retroviruses localisation of viral components (Gag, Pol) to discrete cytoplasmic foci = ?? P-bodies (Crm1p required for export of P-body components) cellular proteins: Crm1p & RNA helicase DDX3

c) + RNA viruses brome mosaic virus HCV (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 2) P-body components required for RNA1-3 replication (membrane-bound complex) WHY? - concentrating genomic RNAs+proteins - promoting interaction with membranes 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 ?

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

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

Thank you for your attention!