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!