Lucie Bartoníčková

ZIB seminar 27th October, 2008

eukaryotic mRNA – subcellular localizations:

- translating mRNA

- mRNA stopped in translation initiation

– mRNA for degradation + translation repressionP-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 granulesaccumulation 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 complexgeneral 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 yeast predominant in mammals

GENE SILENCING

miRNAs

RNA interference

siRNAs

~ 21- 23 nt

RNA-induced silencingcomplex

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

miRNAs siRNAs

translation repressionmRNA 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 virusvirus-like element

studied in

phenotype of mutations

retro-trans-

posons

Ty1 & Ty3 yeast reduced retrotransposition

Ty3 yeast enhanced retrotransposition

+RNA viruses

brome mosaic virus yeastreduced translation & rectruitment

to replication

HCVmammalian cell culture reduced replication

retro-viruses

HIVmammalian 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 particlesmodel: yeast Ty1 (copia-like family) & Ty3 (gypsy-like)

may require P-bodies for life cycle:

pop2Δ (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

miRNAsmay 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!